A STUDY OF ELUTING IgG ANTIBODIES FROM RED BLOOD CELLS: THE

A STUDY OF ELUTING IgG ANTIBODIES FROM RED BLOOD
CELLS: THE COMPARISON FOUR ELUTION TECHNIQUES
KALLAYA KIRDKOUNGAM
A THESIS SUBMITTED IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE
OF MASTER OF SCIENCE
(TRANSFUSION SCIENCE)
FACULTY OF GRADUATE STUDIES
MAHIDOL UNIVERSITY
2008
COPYRIGHT OF MAHIDOL UNIVERSITY
Copyright by Mahidol University

ACKNOWLEDGEMENT
The successful completion of this thesis is credited to the invaluable advice,
supervision and encouragement of the thesis committee.
I would like to express my sincere thankfulness and deepest application to my
major advisor, Dr. Viroje Chongkalwatana, for his kindness, invaluable advice,
guidance, encouragement and support that enabled me to conduct this thesis
successfully.
I also express my appreciation to my co-advisor Assoc. Prof. Sasijit Vejbaesya
for her encouragement, her continual support, understanding thought out my study
and kindness.
I would like to express my kindness thank to CDR Ubonwon Charoonruangrit
for her kindness and helpful.
I would like to express my sincere thank to Dr. Soisaang Phikulsod, Director of
the National Blood Centre, Thai Red Cross Society and the staff of the Antiserum and
Standard Cell Preparation Section and all members of the Red Cell Serology Unit of
the World Health Organization Co-operation Section for their helpfulness and
encouragement during the period of this thesis study.
I also express my appreciation to all members of the Department of Transfusion
Medicine, Faculty of Medicine Siriraj Hospital for their kindness and helpful.
Finally, I am particularly indebted to my loving family and friends. They always
showed understanding and support my work.
Kallaya Kirdkoungam

Fac. of Grad. Studies, Mahidol Univ. Thesis / iv
A STUDY OF ELUTING IgG ANTIBODIES FROM RED BLOOD CELLS: THE
COMPARISON FOUR ELUTION TECHNIQUES.
KALLAYA KIRDKOUNGAM 4837191 SITS/M
M.Sc. (TRANSFUSION SCIENCE)
THESIS ADVISORS: VIROJE CHONGKOLWATANA, M.D.,
SASIJIT VEJBAESYA, Dr. Med. (Transplantation).
ABSTRACT
The objective of this study was to compare in house Acid/EDTA, DiaCidel
Elution Kit, Heat 56 °c and Ether for eluting antibodies from DAT positive red blood
cells. Antibody elution was performed on 175 samples from donors and patients. They
were sensitized with corresponding red blood cells that had were used known antigen
profiles. After that, they were eluted by four techniques of elution. The four eluates
were tested for antibody titration and the titration mean scores were calculated for
paired t-test statistical analysis by SPSS version 13.0.
The results in Rh, Kidd and Diego systems showed that the Ether was the best
method that giving highest the yields and better than other methods. The MNSs
system, for Anti-Mia
In house Acid/EDTA and DiaCidel Elution Kit gave higher
mean scores than Ether but will no statistical significant (p>0.05). In house
Acid/EDTA and DiaCidel Elution Kit were no different in their elution efficiency.
Heat elution gave the lowest yield in every test.
In summary, although Ether was the method that gave the highest yield but it is
very toxic and hazardous for workers in blood bank and for the environment in
general so it is not the best method to use in the laboratory. DiaCidel Elution Kit is
good method but it is very expensive. On the other hand, in house Acid/EDTA has a
very low price but is as good as the DiaCidel elution kit in elution. Therefore, it is a
method that should be considered for use in-house Acid/EDTA in the laboratory.
Currently, blood elution laboratories in Thailand use DiaCidel elution. However,
this method is very expensive. Therefore, it is necessary to seek alternatives methods.
KEY WORDS: ELUTION / DAT / Acid/EDTA
91 pp.

Fac. of Grad. Studies, Mahidol Univ. Thesis / v
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CONTENTS
Page
ACKNOWLEGEDMENT..........................................................................................iii
ABSTRACT……………………………………………………………………….. iv
LIST OF TABLES…………………………………………………………………vii
LIST OF ABBREVIATIONS……………………………………………………..viii
CHAPTER
I INTRODUCTION……………………………………………….………1
II OBJECTIVE……………………………………………………….….…4
III LITERATURE REVIEW………………………………………………..5
IV MATERIALS AND METHODS………………………………………17
V RESULTS………………………………………...……………………24
1. Sample-size Determination for Total Antibody…………...……….24
2. Antibody Titration Sum Scores of Each Elution Method………….25
3. Statistic Analysis………………………………….……………..…31
VI DISCUSSION……………..………………………………………….39
VII CONCLUSION………………….……………………………………46
REFERENCES…………………………………………………………………….47
APPENDIX………………………………………………………………………...51
BIOGRAPHY………………………………………………………………………91

LIST OF TABLES
Table Page
1. A Summery of Practical Methods of Elution…………………….………..14
2. Interpretation of Agglutination Reactions…………………………………22
3. Example of Antibody Titers Scores………………………………………..22
4. Results of Antibody Titration Sum Scores of Each Elution Methods……..27
5. Comparison of Mean Scores among In house, Kit, Heat and Ether by used
SPSS version 13.0…………………………………………………………..32

LIST OF ABBREVIATIONS
Abbreviation Terms
- minus
+ plus
% percent
< less than
= equal to; equals
> more than
less than or equal to
more than or equal to
µl micro liter
Ab. antibody
Ag. antigen
AHG antihuman globulin
BSA bovine serum albumin
°C degree Celsius
DAT direct antiglobulin test
EDTA ethylenediamine-tetraacetate
Eluate liquid end-product of elution
Exp. expiry date
g gram
g gravity force
h hour
HCl hydrochloric acid
HDN hemolytic disease of the newborn
HTR hemolytic transfusion reaction
IgG immunoglobulin G
IgM immunoglobulin M
IAT indirect antiglobulin test

LIST OF ABBREVIATIONS (Continued)
Abbreviation Terms
M molar
min minute
ml milliliter
NaCl sodium chloride
Na2 EDTA disodium ethylenediamine-
tetraacetate
NaN 3 sodium azide
NSS normal saline solution
PBS phosphate buffer saline
pH potential of hydrogen
RBC red blood cell
rpm round per minute
RT room temperature
Temp temperature
TRIS tris(hydroxymethy)
aminomethane
w weak
w/v weight per volume
ix

Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Transfusion Science) / 1
CHAPTER I
INTRODUCTION
The antiglobulin test was discovered by Coombs, Mourant and Race in 1945.It
has been using for detection of weak, non-agglutination or “incomplete” antibodies in
serum. The principle of this test had been described previously in 1908, but it
remained obscure until it was rediscovered by Coombs. More than fifty years after the
development of aniglobulin (AHG) sera, the direct antiglobulin test (DAT) remains
very simple test that investigate the presence of IgG and/or complement components
sensitized red blood cells in vivo that found in both blood donors and patients. If a
patient’s red blood cells test positive with polyspecific AHG sera, monospecific anti-
IgG and anti-complement are used separately to characterize the types of
immunoglobulin sensitizing the red blood cell. The direct antiglobulin test (DAT) was
first used for investigation Rh hemolytic disease of the fetus and newborn (HDFN)
and its application could be used to other antibodies sensitizing red blood cells
detection. Application of the direct antiglobulin test has led to significant discoveries
in human blood groups and has aided in the diagnosis and clinical treatment of many
conditions, such as hemolytic disease of fetus and newborn (HDFN), hemolytic
transfusion reaction (HTR), autoimmune hemolytic anemia (AIHA) and drug-induced
hemolytic anemia. (1)
The direct antiglobulin test positive is antigen-antibody binding or red blood
cells sensitizations depend on ionic, hydrophobic and hydrogen bonds, in addition to
van der waals forces and three-dimensional conformation. There are many reasons
why patients or donors can present with a positive DAT and these include hemolytic
transfusion reactions, autoimmune hemolytic anemia, drug-induced anemia, and
hemolytic disease of newborn (HDN). It may be necessary to remove antibodies those
have sensitized red blood cells in vivo to identify them or red blood cells antigen
phenotype. Lansteiner and Miller were the first pioneers who created the elution
method. After that, several different methods were developed. Antibody elution is

Kallaya Kirdkoungam Introduction/2
attempted to break antigen-antibody binding forces by alterations of ionic strength,
pH, thermal agitation and the use of organic solvents such as heat (56 c), freezing,
acidification, sonication, chaotropic ions, and organic solvents (ether, toluene and
dichoromethane). Because of the heterogeneity of the physical forces involved in
binding, no single elution technique has found universal applicability in the disruption
of all types of antibody-antigen bond (2, 3).
Elution entails the removal of antibodies from sensitized RBCs, as applied in
immunohematology. The main purpose is to recover bound antibodies for
identification of their type by routine serological technique. The results of such
studies are an important part of the investigation of suspected immune-mediated
hemolysis. Elution is also used in combination with absorption method to concentrate
antibodies to detect weakly antibodies expressed RBC antigens, and to purify or
separate antibodies form multispecifics sera. Further, because the sensitivity of a
combined absorption-elution method is hardly ever surpassed by any other serological
test, the results obtained serve to confirm antibody specificity or verify that RBCs are
devoid of a specific blood group antigen. In other studies, elution is undertaken to
remove coating autoantibodies, representation RBCs free of bound immunoglobulins
and, thus, permit accurate phenotyping of other wise autoagglutinating RBCs (4).
A routine method for elution of antibodies sensitized red cell should be quick,
easy and use small amounts of blood sample, effectively and it should give high yield
of all important types of antibody (5). Many methods have been described for the
elution of antibodies from immunoglobin-sensitized red blood cells. Some of these are
commercially obtainable as prepared kits, while others require the preparation of in
house reagents. Unluckily, the choice of an exacting elution method for routine use in
blood banks usually is made individually. Few data are published objectively
comparing the potential of elution methods to elute detectable antibody of different
specificity and sources (6).
In our routine, laboratories use heat elution method which is perfect for elution
of IgM antibodies while only fair for IgG elution. To improve efficiency in routine
work, a commercial elution kit for elute IgG antibodies is better method to consider.
The commercial Acid-glycine/EDTA kit is an absolutely usable method for our
laboratory but expensive; 200 bath per test while the cost of in house Acid-

glycine/EDTA kit is very low; 1 bath/test. The cost per test of the in house is
extremely lower than commercial kit so it should one of the choices.
At the moment, we are finding a better technique than the heat elution to apply
for elute IgG antibodies from positive DAT red blood cells sensitized therefore the
Acid-glycine/EDTA method is one of interested in point of efficiency and cost which
will be compared with the classical, Heat elution, and Ether method.

Kallaya Kirdkoungam Objective/4
CHAPTER II
OBJECTIVES
The purpose of this study is to evaluate the best technique of removing and
recovering bound IgG antibodies from positive DAT red blood cells by comparing
four elution methods: Heat 56 C, Heat-Ether 37 C, In house Acid-glycine/EDTA and
commercial acid-glycine/EDTA kit (DiaCidel Elution Kits, DiaMed).

Fac. of Grad. Studies, Mahidol Univ. M.Sc. (Transfusion Science) /5
CHAPTER III
LITERATURE REVIEW
EARLY HISTORY OF BLOOD TRANSFUSION
Ancient man had different belief in blood and blood transfusion. The tribes of
Central Australia had custom which a sick old man drunk the blood of young man. In
South American, there was the method that most popular of driving out a bad spirit
was by vein section in the belief that the demons escaped with blood. The Egyptians
anointed heads with oil mixing blood to treat graying hair and baldness. In the
gladiatorial arena of the Roman Empire, they also used blood of dying to bathed and
drunk; they were said this was a method to have rushed courage of the gladiators. The
Greek believed that the blood which was supposed to be manufactured by the liver
and wine was considered as contributing greatly to its formation. The first recorded
transfusion was performed in 1490 at Rome on Pope Innocent VIII who lay dying of
old age because was proposed injection the blood from three young, healthy boys into
his veins. Blood Transfusion experimental progressed step by step, initial transfusions
used blood from animals transfer to man. Using the blood of an animal was the
dominating idea of the time in the belief that the characteristic of the animal would be
engrafted upon the human recipient and perhaps a criticism of mankind is implied in
the usual choice of lamb’s blood. The patient was invariably bled before he was
transfused, the purpose being to remove bad blood to let in good. (7)
The first experiment happened in 1666 at Oxford University by William Harvey.
His experiment showed that intravenous injection substances into animals could exert
a systemic effect. Richard Lower (1631-1703) demonstrated that blood become red
after pass lungs and in 1666 he showed experiment of blood transfusion from one dog
to another. (8) In 1818, James Blundell was an obstetrician at Guy’s and St. Thomas
hospital in London, successfully carried out blood transfusion in postpartum
hemorrhage female patients. He developed a syringe with a two-way stopcock and

Kallaya Kirdkoungam Literature Review /6
this was used with considerable degree of success to treat women patients. He was the
first scientist confirmed that transfusion blood from one human to another is the
correctly way. This represented the beginning of the modern era of transfusion
medicine. Following Blundell, several therapeutic cases with blood transfusion were
recorded. (8, 9) However, blood transfusion without knowledge of blood group
system was very dangerous, ABO incompatibility made patients died and blood
transfusion in different species was very mistake because incompatibility of blood
between species often resulted in lysis in 2 minutes.(9)
MODREN HISTORY OF BLOOD TRANSFUSION
DISCOVERY OF THE ABO BLOOD GROUP SYSTEM
The important man in modern blood transfusion history is Karl Landsteiner
(1818-1943) who discovered ABO blood group system in 1901 and he awarded Nobel
Prize for Medicine in 1930. His publication in 1901, he descried the reactions
between the red cell of other and he realized that this was an immunological basis
phenomena. In order to explain the clumping patterns, Lansteiner postulated that there
were two antigens (A and B) and two antibodies against those antigens (Anti-A and
Anti-B). He assumed the presence of the antibodies in the sera of individuals who did
not express those antigens, which later named Lansteiner’s Law. Initially, he was
classified only three blood groups which he named A, B and C. Serum from group C
subjects clumped the cell of those from groups A and B. (8, 10)
The next year, Decastello and Sturl, two of Lansteiner’s pupils in Vienna,
confirmed his job in a larger study of 155 individuals and also identified four subjects
(2.5%) with no agglutinins in their own serum but whose red cells were agglutinated
by serum from subjects with all of the three previously identified blood groups (group
AB).(8)
Lansteiner’s Law was an important step toward the safe practice of blood
transfusion, where transfusion should be performed between individuals whose blood
components would not agglutinate upon mixing. It was reasonable to assume that the
hemaagglutination due to mismatch would also occur inside the body if it occurs in

the test tube. Therefore, ABO typing before any transfusion was logical. To
crossmatch also was wise because unknown antigens or antibodies could be present.
(10)
DISCOVERY OF THE Rh AND OTHERS BLOOD GROUP SYSTEM
More than 50 years ago after the discovery of the Indirect Antiglobulin Test
(IAT) that could be applied to the blood group antibodies detection. There was rapid
increase in the identification of alloantibodies that caused transfusion reactions or
Hemolytic Disease of newborn (HDN).After Rh, antibodies in the Kell, Duffy, and
Kidd blood group systems were the next in clinically significant antibodies to be
revealed. (11) The Rh blood group system is considered the most polymorphic human
blood group system because it consists of at least 45 independent antigens. The Rh
antigens have the great immunogenicity so this system is considered its clinical
important in transfusion medicine and are involved in hemolytic transfusion reactions,
hemolytic disease of the newborn (HDN) and autoimmune hemolytic anemia. The Rh
system is so named because the antibodies made in 1940 by Lansteiner and wiener in
rabbits (and later guinea pig) in response to injection of Rhesus monkey (Macacus
rhesus) RBCs were thought to be of the same specificity as the human antibody
investigated by Levine and Stetson in1939 following a transfusion reaction. Some
year later it was recognized that the animal and human Rh antibodies did not react
with the same antigen. However, the buildup of thousands of publications made it
impossible to change the name of the clinically important human antibody form anti-
Rh. The original human specificity is now known as anti-D (in the Fisher-Race
notation) or anti-Rh0 (in Weiner’s notation). Not long after the discovery of the
original Rh antigen, i.e., the D antigen, antibodies were identified that detected
antigens related to D but which were not the same as D. By 1945, the five major
antigens of the Rh system (D, C, E, c, e) were known and interpretation of serologic
results grew in complexity. (12)
The Kell blood group system was discoveries in 1946, just a few weeks after
the introduction of the antiglobulin test. The RBCs of a newborn baby, thought to be
suffering from Hemolytic Disease of newborn (HDN), gave a positive reaction in the

Direct Antiglobulin test positive (DAT). The serum of the mother reacted with RBCs
from her husband and her older child and later was shown to react with 9 percent of
random donors. The system was named from Kellecher, her surname, and the antigen
is referred to as K (synonyms: Kell, K1). Three years later the antithetical antigen, k
(synonyms: Cellano, K2), which is of high incidence in all populations, was identified
by typing large numbers of RBC samples with an antibody that had also caused mild
HDN. The Kell system remained a simple two-antigen system until 1957, when the
antithetical Kpa
and Kpb
antigens and the K0 (Kellnull) phenotype were reported.
Subsequently, the number of Kell antigens has grown to 24 making Kell the most
polymorphic blood group system known. (11)
The Kidd blood group is a major antigenic system in human red blood cells, and
this antigens system is defined by two antithetical specificities, Jka
and Jkb
, and a third
rare recessive gene, Jk, that produces neither Jka
nor Jkb
antigens. The Kidd antigens
are localized on a 43 kDa red blood cell integral membrane protein that functions as
urea transporter. This blood group system was discovered by Allen et al. in 1951 in
mother who had no history of transfusion, and the women had given birth to an infant
with mild hemolytic disease that was because of anti-, Jka
. The antithetical ant-Jkb
was reported by Plaut et al. in 1953. Anti-Jkb
can cause hemolytic disease that is
uaually a mild and has a benign prediction. It is clinically significant since Jk
antibodies can cause acute and delayed transfusion reactions as well as HDN. (13)
The Duffy (Fya
) blood group antigen was first reported in 1950 by Cutbush Et
al, who described the reactivity of an antibodies found in a hemophiliac male that
usually transfused. This blood group system bears the patient’s surname, Duffy, and
the last two letters provide the shortened nomenclature (Fy). Fyb
was found one year
later. In 1975, Fy was identified as the receptor for the malarial parasite Plasmodium
vivax. This discovery explained the predominance of the Fy(a-b-) phenotype (Fynull),
which confers resistance to malarial invasion, in Blacks originating from West
Africa.(11)

DISCOVERY OF THE ANTIGLOBULIN TEST
In the 1940s, the actual nature of antibodies was still unknown, but seemed to be
associated with the serum globulins. Race, Mourant and Weiner concluded that there
were two types of Rh antibody: one that bound to the RBC surface and caused
agglutination (the “complete” antibody) and another that absorbed to the RBC surface
but did not cause agglutination (the “incomplete” antibody).(14)
In 1945, Coombs, Mourant and Race described technique for detecting
attachment of Rh antibodies in serum that did not produce agglutination. This test
is know as the antiglobulin test (AHG) and uses antibody to human globulin. In
1946, Coombs and associates described the use of AHG to detect in vivo sensitization
of the red cells of babies suffering from hemolytic disease of the newborn (HDN).
Although the test was initially of great discovery in the investigation of Rh hemolytic
disease of the newborn, it was not long before its many for detection of other IgG
blood group antibodies became clearly. The first of the Kell blood group system
antibodies and its associated antigen were reported only weeks after Coombs had
described the test. (14, 15)
The principle of the instrumental in introducing the antiglobulin test to blood
group serology had in fact been described by Moreschi in 1908 before Coombs and
associates. The study of Morechi involved the use of rabbit antigoat serum to
agglutinate rabbit red cells, which were sensitized with low no agglutinating does of
goat antirabbit red cells serum. Coombs’s production involved the injection rabbits
with human serum to produce antihuman serum. The absorption is used to remove
heterospecific antibodies and the dilution to avoid prozone but the antiglobulin serum
still retained sufficient antibody activity to permit cross-linking of adjacent red cells
coated with IgG antibodies. (15)
The antiglobulin test was first used to demonstrate antibody in serum, but later
the same principle was used to demonstrate in-vivo sensitizing of red cells with
antibodies or complement components. As used in immunohematology, antiglobulin
testing generates visible agglutination of sensitized red cells. An indirect antiglobulin
test is used to demonstrate in-vitro reactions between red cells and antibodies that
sensitize, but do not agglutinate, cells that express the corresponding antigen. (16)

THE ANTIGLOBULIN REACTION
Pentameric IgM antibodies are able to bridge the gap between adjacent red cells
and cause direct agglutination; IgG antibodies are not able to do this. The detection of
ABO IgM antibodies using simple agglutination reactions is the basis of ABO typing
and cross-matching to confirm ABO incompatibility. In 1944, when Robin coombs
was researching in the Pathology Laboratories in Cambridge, it had only just been
realized that antibodies resided in the globulin fraction of serum. Some antibodies
would bind to red cells without causing agglutination as the cells were show to carry a
globulin on their surface even after being washed several times. These were referred
to as incomplete antibodies. What was needed was a reagent that bridged the gap
between antibody-sensitised red cells. It was found that the bound globulin could be
recognized by an antiserum against globulin prepared in a suitable animal. In the case
of Rh D testing, the antiglobulin serum was usually prepared by used human globulin
immunizing rabbits or goats. This reagent then cause lattice formation between
adjacent red cells and build up red cells visible agglutination. The simplest application
of the antiglobulin technique was to test patient’s cells to determine if they carried
antibodies on their surface for example in the case immune hemolytic anemia or in
patients who often blood transfusion that the donor red blood cells in their circulation
were sensitized. This was done by incubating washed red cells from the patients with
antiglobulin reagent-agglutination indicating sensitization- and this is the direct
antiglobulin test. This test could be adapted to screen for the presence of red cell-
reactive antibodies in serum for example when testing patients who were receive
transfusion. Serum was reacted with test red cells- perhaps from units of blood being
tested for compatibility previous to transfusion. After several washes the antiglobulin
reagent was added to see agglutination if the red cells had become sensitized. This is
the indirect antiglobulin test. (17)

ANTIHUMANGLOBULIN REAGENTS
AHG, originally known as Coombs’ serum, is produced by injecting human
globulin into laboratory animal e.g. rabbits or sheep or goat and refining the resultant
immune serum to remove unwanted agglutinins. In the early development of
antiglobulin reagents, standard for manufacture, stability, potency, and specificity,
had not been established. The sensitivity and concentration of anti-IgG and
anticomplement (C3b, C3d) in polyspecific AHG sera varied among batches and
manufactures. Researchers such as Garratty and Petz, Howard et al, and Issitt,
investigated various sources of sera and made numerous recommendations regarding
the level of anti-IgG and anticomplement in polyspecific AHG used in the direct
antiglobulin positive (DAT) and indirect antiglobulin test (IAT). (1)
Several AHG reagents have been defined by the Food and Drug Administration
(FDA) Center for Biologics Evaluation and Research (CBER). There are two major
types of antihuman globulins, Polyspecific AHG and Monospacific AHG.
Polyspecific AHG contains antibody to human IgG and to the C3d component of
human complement. Other anti-complement antibodies such as anti-C3b, anti-C4b, or
anti C4d may be also present. Commercially prepared polyspecific AHG contains
little, if any, activity against IgA and IgM heavy chains. However, the polyspecific
mixture may contain antibody activity to kappa and lambda light chains common to
all immunoglobulin classes, thus reacting with IgA or IgM molecules. Monospecific
AHG reagents contain only one antibody specific: either anti-IgG or antibody to
specific complement components such as C3b or C3d. Licensed monospecific AHG
reagents in common use are anti-IgG and anti-C3b-C3d. Reagents labeled “anti-IgG”
contain no anticomplement activity. Anti-IgG reagents contain antibodies specific for
the Fc fragment of the gamma heavy chain of the IgG molecule. If not labeled
“gamma heavy-chain specific” anti-IgG may contain anti-light-chain specificity and
therefore react with red blood cells sensitized with IgM and IgA as well as with IgG.
Anti-complement reagents such as anti-C3b-C3d reagents are reactive against the
designated complement components only and contain no activity against
humanimmunoglobulins. (15)

DIRECT ANTIGLOBULIN POSITIVE (DAT) AND ITS SIGNIFICANT
After the development of antihumanglobulin (AHG) sera, the direct antiglobulin
test (DAT) remains simplest test that demonstrate that presence of IgG and/or
complement components sensitizing red blood cells in vivo. If a patient’s red cells test
positive with polyspecific AHG sera, monospecific anti-IgG and anti-complement are
used separately to characterize the class of immunoglobulin sensitizing the red blood
cell. When investigating serologic results of the DAT, blood banker collect pertinent
information such as the patient’s history had been transfusions, pregnancies,
diagnosis, and medication and use routine pathways or rules for testing and evaluation
to develop conclusion. In most case, this approach leads to the correct interpretation
of most serologic problems. However, often a patient presents with complex serologic
result that do not seem to correlate with clinical findings. The blood banker is then
required to reassess the initial interpretation and approach the problem in a more
innovative way such as elution and absorption. (1)
DISCOVERY OF ELUTION TECHNIQUE
The first antibodies coating RBCs elution technique for cold antibodies is the
heat elution discovered by Landsteiner and Miller as early as 1902. Landsteiner was
also instrumental in developing the second technique for eluting antibodies from
RBCs. In co-operation with van der Scheer, he created a method for dissociating
azostromato-antibody complexes. This method was modified by Kidd to demonstrate
incomplete IgG antibody on the RBCs of acquired hemolytic anemia patients. Elution
method of Kidd that hemolysed RBCs with ten times their volume of distilled water.
The precipitation of stroma with 1 N HCl until the pH was in range of 5.6 to 5.8 and
later the stroma was eluted of antibody using a citrate HCl buffer pH 3.2 to 3.4, an
amount of buffer equal to twice the volume of packed stroma was used and
subsequent neutralization of elutes with 5 N NaOH (3,6). Greenbelt who used a
c of toluene and distilled water to precipitate stroma, then, harvested it
using glass wool used for filtration and recovered bound antibodies by elution at 56

into either saline or albumin (18). The freeze-thaw is Weiner’s method that
destroys RBCs, 50% cold ethanol used for precipitating the stroma and recovering
antibody from the precipitate stroma with saline 37°C (19).
For testing IgM antibodies eluates, hemoglobin-free elute are important but
they are not essential for indirect antiglobulin tests (IAT) (3). For this objective, Harry
Rubin created the ether elution method at 37°C for warm antibodies; in addition some
observations were made on the sensitized red blood cells of patients with autoimmune
hemolytic anemia (20).
An elution procedure that is used routine must give a best yield of all clinical
significant antibodies, must be appropriate to small amounts of blood sample, quick,
simple and not be hazardous to blood banker. The method of Landsteiner and van der
Scheer does not fulfill these standards. The results are unsure and regularly
reproducible. Such preparations are always grossly contaminated with hemoglobin
that interfere will testing of eluate. Kidd’s method give high yields and elute is clear
and colorless but used a long time and requires steps of varying complication to
separate the stomata of the red blood cells and extract the antibodies by adjust of pH.
Relatively large volumes of blood sample are needed for the stoma techniques (20,
18). Rubin’s ether elution method is dangerous for the blood bank worker. Ether is
highly flammable and must be strictly regulated in regard to its use and storage (21).
Numerous methods were created from 1977 to 1982. (Table1.) Jenkins and
Moore used 0.8 mol/l phosphate buffer pH 8.2 neutralize eluates prepared by the
glycine-HCl method of Kochwa and Rosenfield. Rekvig and Hannestad and Bush
created glycin-HCl elution method for use with intact RBCs instead of stroma. And
these two creations have been developed to modern commercial elution kits such as
Elu- Kit II, Gamma Biological Inc. Houston, TX and DiaCidel Elution Kit, DiaMed
AG, Switzerland (3). Another elution method was described by Chan-Shu and Blair,
by Bueno R. al using xylene that elution technique was superior to methods using
ether, digitonin-acid and heat. Ellisor et al compared five elution methods including
heat elution (56 c), heat elution (45 c), Rubin’s ether elution method, Marsh’s
adapted ether elution method and digitonin-acid elution was first described by

Kochwa s., Rosenfield R.E. They found that the Marsh’s adapted ether elution method
gave the highest yield of antibody activity the greatest percentage of times (21).
In recent years, various methods of antibody dissociation have been developed
that do not destroy the RBCs. The objective is to remove either IgM or IgG
autoantibody in a way that permits accurate phenotyping of the RBCs. Three methods
have been developed to permit phenotyping of IgG-coated RBCs with reagent antisera
require using by the Indirect Antihumanglobulin test. The first, Edward et al have
Investigated the quinoline derivative choloquine diphosphate (200 mg/ml, pH 5.0) to
dissociate antibodies without denatured red cell antigens. They found the choloquine
dissociation technique to be of value in the examination of red blood cells with a
positive DAT, either or the qualitative or quantitative expression of antigen. The
second, other investigators studied the effect of acidic ethylenediaminetetraacetic acid
(EDTA)-glycine mixtures to remove IgG from RBCs without destroying RBC
antigens. The third, Caruccuo L. et al found that the formamide method was efficient
in removing antibodies from RBCs. The patient samples with a positive DAT had
antibodies recovered with the same specificity when compared to the acid-based
technique. The preparation time length was similar for both formamide and acid-
based methods (2, 3, and 22).
Table 1. A Summery of Practical Methods of Elution (2)
From intact RBCs: Author(s) Elution Restoration*
Landsteiner & Miller 56ºC
Rubin Ether at 37ºC Evaporation at 37ºC
Rekvig & Hannestad, Bush Cold glycine-saline PH 8.2 PO4 buffer
Eicher et al Freeze (-20 to -70ºC);rapid thaw
Branch et al Chloroform at 56ºC Centrifugation
Massuet et al Chloroform/trichlorethylene at 37ºC Centrifugation
Deisting et al D-limonene at 56ºC Centrifugation
Ellisor et al Dichlormethane Evaporation at 56ºC
Chan-Shu & Blair, Bueno et al Xylene at 56ºC Centrifugation
Bird & Wingham, Jimerfield Sonication
Meier et al Microwaves

From red cell stroma:
Author(s)
Stroma Prepparation Elution Restoration*
Kidd Freeze-thaw/HCl Citrate-HCl NaOH
Kominos & Rosenthal Freeze-thaw/HCl 8% NaCl Dialysis
Vos & Kelsall Freeze-thaw/HCl Ether Evaporation
Greenwalt Toluene/H2O Albumin/saline at 56ºC
Weiner Freeze-thaw,50%ethanol Saline at 37ºC
Kochwa & Rosenfield Digitonin Glycine-HCl Dialysis
Jenkins & Moore Digitonin Glycine-HCl PH 8.2 PO4
buffer
To prepare antibody-free red blood cells for
phenotyping: Author(s)
Eluant
Reid Dithiothreitol,2-mercaptoethanol
Edward et al Chloroquine diphosphate
Louie et al,Kosanke et al EDTA-glycine HCl
*Method used to remove eluant or restore eluant tonicity/neutrality
= stroma precipitant
ELUTION AND ITS SIGNIFICANT
In cases of hemolytic disease of the newborn (HDN) or hemolytic transfusion
reactions, specific antibody (or antibodies) is usually detected in the eluate. Usually
the same specificity can be detected in the patient’s (or, in HDN, the mother’s) serum,
although eluates may help in antibody identification when serum reactions are weak.
When the eluate reacts with all cells tested, autoantibody is the most likely
explanation, especially if the patient has not been recently transfused. When no
unexpected antibodies are present in the serum, and if the patient has not been
recently transfused, no further serologic testing of an isolated autoantibody is
necessary.
Sometimes no reactivity is detected in the eluate, despite reactivity of the cells
with specific anti-IgG. The cause may be that the eluate was not tested against cells
positive for the corresponding antigen, notably group A or group B cells. Antigens of

low incidence are also absent from most reagent cell panels. It may be appropriate to
test the eluate against red cells from recently transfused donor units, which could have
stimulated an alloantibody to a rare antigen, or, in HDN, against cells from the father,
from whom the infant may have inherited a rare gene.
Reactivity of eluates can be enhanced by testing against enzyme-treated cells or
by the use of solid-phase or other enhancement techniques, such as polyethylene
glycol (PEG). Antibody reactivity can be increased by the use of a concentrated
eluate, either by alteration of the fluid-to-cell ratio or by use of commercial
concentration devices. Washing the red cells with low ionic strength saline (LISS),
instead of normal saline, may present the loss of antibody while the cells are being
prepared for elution.
Certain elution methods give poor results with certain antibodies. When eluates
are no reactive yet clinical signs of red cell destruction are present, elution by a
different method may be helpful. If both serum and elute are no reactive at all test
phases, and if the patient has received high-dose intravenous penicillin or other drug
therapy, testing to demonstrate drug-related antibodies should be considered. Patients
may have a positive DAT and no reactive eluate with any evidence of hemolysis, and
exhaustive pursuit f an explanation is not usually indicated. (23)

CHAPTER IV
MATERIALS AND METHODS
STUDY DESIGN: Experimental controlled trial
1. DAT negative groups O red blood cells were obtained from the ACD
anticoagulant specimens collected by National Blood Center, Thai Red
Cross Society. They had already been typed for a complete antigenic
profile, e.g., Rh system, MNSs, Lewis, P1, Duffy, Kidd, Kell and Diego.
These red blood cells will be used for in vitro sensitization. (Example for
complete antigenic profile red blood cell in Appendix.)
2. Antibodies know type from donors and patients were selected and used for
in vitro sensitization. Each antibody will be titrated before in vitro
sensitization.
3. Prepared in vitro sensitization RBC using Individual RBC from 1 (4 ml)
and antibody known type from 2 (4 ml) and selected the one those gave
DCT 1+ to 2+ (grade) or 2 to 8 (score).
4. Eluted 1 ml of the in vitro sensitized RBC by 4 techniques; heat, ether, in
house acid-glycine/EDTA and the commercial DiaCidel elution kit
(DiaMed AG, Switzerland) those gave 1 ml of each elution returned.
5. The eluates were titrated.
6. Comparison the results.

Kallaya Kirdkoungam Materials and Methods /18
Flow Chart of Study Design
MATERIALS
1. Samples
1.1 Group O DAT negative RBC with known antigenic phenotype.
2.2 Antibodies-identified antiserum 175 samples from donors and patients, e.g.,
20 anti-D/ 2 anti-C/ 21 anti-E/ 30 anti-Mia
/ 2 anti-c/ 7 anti-Fya
/ 5 anti-Fyb
/ 10 anti-Jka
/
10 anti-Jkb
/1 anti-S/ 8 anti-E+ Mia
/ 9 anti-E+ Jka
/ 5 anti-S+Mia
/10 anti-E+c/ 5 anti-
C+e/ 5 anti-Dia
/ 11/anti-E+c+Mia
/ 10 anti-E+c+Jka
/1 anti-E+S+Dia
/1 anti-Mia
+Jkb
/ 1
anti-E+Mia
+Jka
/ and 1 anti-S+Jka
.
In vitro sensitized
RBCs group O DAT positive
Heat Ether Acid-glycine/EDTA
(DiaCidel kit)
Eluate titration
Acid-glycine/EDTA
(In house)

2. Reagents
2.1 , ), Diethyl ether and Bovine
Albumin 30%
2.2 In house acid-glycine/EDTA
2.2.1 EDTA 10%
Disodium ethylenediamine-tetraacetate (Na2 EDTA) 2 g makes
up to 20 ml with distilled water.
2.2.2 Glycine-HCl buffer (pH 1.5) 0.1 M
Glycine 0.75 g makes up to 100 ml with sodium chloride 0.9%
and adjusts to pH 1.5 using concentrated hydrochloric acid
(HCl).
2.2.3 TRIS-NaCl 0.1 M
Tris(hydroxymethy)aminomethane (TRIS) 12.1 g and sodium
chloride 5.25 g makes up to 100 ml with distilled water.
2.3 DiaCidel Elution Kit (DiaMed AG, Switzerland)
2.3.1 DiaCidel, Wash solution (concentrated) containing
Glycine-NaCl buffer, in 30 ml vials. Preservative: 1.0-% NaN
3.
2.3.2 DiaCidel, Elution solution containing a low pH glycine buffer
with color indicator, ready-for-use, in 10 ml vials.
2.3.3 DiaCidel, Buffer solution containing Tris buffer with bovine
albumin (1.2%), ready-for-use, in 10 ml vials. Preservative:
0.1% NaN3.
3. Equipments
3.1 Sero-fuge centrifuge 3,000 rpm
3.2 Water bath 37 °c
3.3 Fume Hood
3.4 Test tube 13x100-mm, 10x775-mm and 12x75-mm
3.5 Auto pipette 100-1000 µl and 50-100 µl

METHODS
1. In Vitro Sensitized Red Blood Cells
1.1 Washed group O DAT negative red blood cells three times in 0.9% normal
saline and completely decant saline.
1.2 The red blood cells have antigens that corresponding human derived 175
sample antibodies those were obtained from patients or donors. Sensitized
175 pairs of antigen-antibody at 37 c for 30-60 minute in water bath.
1.3 Washed 3 ml of the sensitized red blood cells 4 times with normal saline
and washed 1 ml 4 times with DiaCidel wash solution, the last washed
supernatant was harvested for parallel testing for compare the elute.
1.4 The effectiveness of red blood cells coating antibodies was confirmed by
conventional tube test direct antiglobulin test and their grading between 0
to 4+ or their scoring 0 to 12 positive.
1.5 Sensitized red blood cells were eluted by use four techniques of elution.
2. Method for Elution
2.1 Heat Elution (24)
2.1.1 Bovine albumin 6% was prepared by diluting 30% bovine albumin
with saline. Washed 1 ml of the sensitized red blood cells 4 times
with normal saline. The final wash supernatant was kept for
parallel test.
2.1.2 Mix 1 ml of the sensitized red blood cells to 1 ml dilute bovine
albumin in a 13x100-mm test tube.
2.1.3 Then incubated the tube at 56 c for 10 minutes with agitating
periodically.
2.1.4 Centrifuged the tube at 1000xg for 2 minutes.
2.1.5 Immediately transferred the supernatant eluate into a clean test
tube.

2.2 Ether Elution (24)
2.2.1 Diethyl ether reagent grade or anesthesiology grade.
2.2.2 Washed 1 ml of the sensitized red blood cells 4 times with
normal saline.
2.2.3 Mixed 1 ml of the sensitized red blood cells, 0.5 ml of saline
and 1 ml of ether together in a 13x100-mm test tube.
2.2.4 Stopped the tube with a cork and agitated the tube vigorously for 1
to 2 minutes.
2.2.5 Then incubated at 37 c for 15 minutes.
2.2.6 Centrifuged the tube at 1000xg for 5 minutes.
2.2.7 Carefully removed the cork to release pressure slowly removed
and discarded the upper layer ether.
2.2.8 Transferred the hemoglobin-stained eluate below the stromal layer
into a clean 13x100-mm test tube.
2.2.9 Using a Pasteur pipette, periodically bubble air through eluate,
until it no longer smelled of ether.
2.3 DiaCidel Elution Kit
2.3.1 Added 1 ml of washed sensitized red blood cell and 1 ml of
DiaCidel elutes solution together and mixed well.
2.3.2 Centrifuged immediately at 900 g (3000 rpm) for 1 minute.
2.3.3 Transferred eluate to clean tube.
2.3.4 Added 5 drops (250 l) of DiaCidel buffer solution to eluate and
mixed well. Observed the forming of a blue color, indicating that
neutral pH 6.5-7.5 is reached. If the blue was not obtained,
added more buffer (1 drop (50 l) at a time) while mixing.
2.3.5 Eluate was now ready for testing.
(Reference from DiaCidel for acid-elution of Serological antibodies:
Product-Identification: 45630)

2.4 In House Acid-glycine/EDTA (25)
2.4.1 Mixed 1 ml of 0.1 M glycine-HCl buffer (pH 1.5) to 250 l of
10% EDTA in a test tube. This would be the ELUTION
REAGENT.
2.4.2 Placed the 1 ml of washed sensitized red blood cell into
a 12x75- glass tube.
2.4.3 Added 1 ml of ELUTION REAGENT to the sensitized red blood
cell, mix well, and placed on room temperature (22-24 °c) for 1
minute (caution: over time will cause irreversible damage to the
red blood cell)
2.4.4 Added 140 l of 1 M TRIS-NaCl, mixed, and immediately
centrifuged at 1,000xg for 1 minute.
2.4.5 Removed supernatant (eluate) in to tube test.
2.4.6 Carefully adjusted pH of the eluate between 7.0-7.4 using 1 M
TRIS and pH paper, (caution: 1 M TRIS is a strongly alkaline
agent, so only very small amount is required for adjusting pH).
3. Eluate Titration (26)
3.1 Serial two fold dilution of the eluate e.g. undiluted, 1:2, 1:4, 1:8, 1:16,
1:32, 1:64, 1:128, 1:256 and 1:512.
3.2 Using separate pipettes for each dilution transfer 50 µl of each diluted sera
into the appropriately test tubes, and added 25 µl of the red cell
suspension. (Red blood cells were the same one that was used in vitro
sensitized for this antiserum.) Mix well, and incubated at 37 °c 1 hour.
3.3 Washed 3 times with NSS. The last wash discarded NSS absolutely.
After that added 100 µl Coombs serum on dry drop of pack red cells. Mix
well.
3.4 Immediately spin, 3,000 rpm 15 second.
3.5 Interpretation of agglutination reactions and titration sum scores (Table 2
and Table 3)
3.6 Titration sum scores of all eluates show on Table 4.

Table 2. Red cell antigen-antibody reactions: serologic grading. (26)
Table 3. Example of Antibody Titers Scores (26)
Reciprocal of Serum DilutionSample No.
n 2 4 8 16 32 64 128 256 512 Sum
Grade 3+ 3+ 3+ 2+ 2+ 2+ 1+ + + 0 noneSample 1
Score 10 10 10 8 8 8 5 3 2 0 64
Grade 4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+ + 0 noneSample 2
Score 12 12 12 10 10 8 8 5 3 0 80
Grade 1+ 1+ 1+ 1+ + + + + + 0 noneSample 3
Score 5 5 5 5 3 3 3 2 2 0 33
Statistical Analyses
Statistical analyses were performed using a commercially available software
package (SPSS version 13.0 for windows). The Two-sample paired t-test was used to
compare sum scores of the antibody titration results that obtained from Heat, Ether,
In-house Acid/EDTA and DiaCidel Elution Kit. The mean scores of antibody titration
were used to compare to the efficiency potential of two elution methods. A level of P
0.05 was considered as statistical significant.
The study was approved by the Ethical Committee of Faculty of Medicine Siriraj
Hospital, Mahidol University (No.004/2550).
Macroscopically-observed Findings Grade Score
One solid agglutinate 4+ 11-12
Several large agglutinates, clear background 3+ 9-10
Medium-size agglutinates, clear background 2+ 6-8
Small agglutinates, tiny agglutinates turbid background 1+ 5
Very small agglutinates, turbid background 1w
3-4
Barely visible agglutination, turbid background ½ or trace 1-2
No agglutination 0 0

Kallaya Kirdkoungam Results/24
CHAPTER V
RESULTS
1. Sample-size Determination for Total Antibody
To estimate the required samples from population proportion using inverse
formulation, the calculated value can be accomplished after considered the extent of
, , and as show below
N/group = [ (Z /2 + Z ) ] 2
______________
2
= Type I error = 0.05 (2-sided), Z0.025 = 1.96
= Type II error = 0.2 (1-sided), Z0.2 = 0.842
= Standard deviation (SD) of difference in total score between 2
methods
d = Difference in mean of scores between 2 methods
For the value of and , there are two independent ways used select; (1) from
the previous study with the most similar of population and designed experimental and
(2) if no previous study, and value can obtained by pilot study. In this study, the
investigation of Nathalang O., Bejrachadra S. (27) is selected for and value. The
is standard deviation (SD) of difference in total score (from 10 dilution) between 2
methods (Heat & In house Acid/EDTA), (Heat & DiaCidel Kit), (Heat & Ether), (In
house Acid/EDTA & DiaCidel Kit), (In house Acid/EDTA & Ether) and (DiaCidel
Kit & Ether).
Using this equation, Z /2 = 1.96, Z = 0.842, = 14.25, = 3.05, this is

N/group = [(1.96 + 0.842) (14.25)] 2
__________________
(3.05)2
= 171.348
Thus, for complete answer to the research question, the size of appropriate
sample must not less than 172.
2. Antibody Titration Sum Scores of Each Elution Method
As show in Table 4, sample no. 1 to sample no. 113 are single antibody,
sample no. 114 to sample no.152 are the combination of two antibodies and sample
no. 153 to sample no. 175 are combined of three antibodies. Sample no. 25 and 26 are
equal scores in DAT (5) and sum scores of titration before sensitized (14) but much
different in sum scores titration of In house method eluate because red blood cells for
In vitro sensitization are not same. Antigens of each red blood cell are different so
eluates titration sum scores are different.
Before sensitization, antibodies from donors or patients were tested for
titration; sum scores show in column Ab (Table 4 page 27 to 31). Sensitized red cells
positive DAT were scoring and interpreted (according to Table 2 page 22) from 2 to
12. Next step, they were eluted by Heat, In House Acid/EDTA, DiaCidel Kit and
Ether. The supernatant eluates were tested for antibody titration and every titration
sum scores show in the columns of sum scores of titration were used to calculate in
paired t-test by SPSS. Last washed supernatant of DAT cells were negative control
for each method and Combs Control cells (C.C.C.) were confirmed that they were true
negative.

Table 4. Results of Antibody Titration Sum Scores of Each Elution Methods
Sum Scores of TitrationSample
No.
Anti From DAT
Ab In House Kit Heat Ether
Negative
Control
C.C.C
1 D Donor 4 38 26 18 21 39 0 +
2 D Donor 5 66 33 39 9 51 0 +
3 D Donor 5 68 43 38 25 53 0 +
4 D Donor 3 53 27 20 15 38 0 +
5 D Donor 4 42 33 28 18 39 0 +
6 D Donor 3 67 19 19 6 27 0 +
7 D Donor 6 44 42 42 33 47 0 +
8 D Donor 4 59 32 32 23 40 0 +
9 D Donor 4 55 40 37 24 36 0 +
10 D Donor 7 80 55 46 26 67 0 +
11 D Donor 5 72 48 39 19 57 0 +
12 D Donor 5 85 37 45 8 53 0 +
13 D Donor 2 14 15 15 3 16 0 +
14 D Donor 2 45 12 12 7 12 0 +
15 D Donor 3 36 18 16 12 32 0 +
16 D Donor 3 47 25 22 9 35 0 +
17 D Donor 3 44 15 15 5 19 0 +
18 D Donor 4 75 21 20 9 37 0 +
19 D Donor 3 73 14 13 9 27 0 +
20 D Donor 3 57 12 12 5 18 0 +
21 C Patient 4 29 8 12 2 12 0 +
22 C Patient 2 9 6 8 3 8 0 +
23 E Donor 2 20 9 12 11 18 0 +
24 E Donor 4 14 14 10 9 33 0 +
25 E Donor 5 14 2 2 2 18 0 +
26 E Donor 5 14 14 9 2 28 0 +
27 E Donor 6 35 25 14 32 39 0 +
28 E Donor 4 22 9 14 8 21 0 +
29 E Donor 3 18 5 5 5 13 0 +
30 E Donor 5 30 35 24 9 38 0 +
31 E Donor 4 36 20 15 7 32 0 +
32 E Donor 2 12 7 4 3 12 0 +
33 E Donor 8 33 27 18 27 52 0 +
34 E Donor 4 32 9 12 2 14 0 +
35 E Donor 5 32 14 13 4 38 0 +
36 E Donor 3 12 2 2 2 5 0 +

No.
Anti From DAT
Negative
Control
C.C.C
37 E Donor 8 43 23 36 13 40 0 +
38 E Donor 3 14 7 5 9 22 0 +
39 E Donor 7 38 20 21 14 32 0 +
40 E Patient 7 47 17 17 6 30 0 +
41 E Patient 3 37 16 10 2 25 0 +
42 E Patient 3 22 6 6 2 11 0 +
43 E Patient 2 16 2 2 2 3 0 +
44 c Donor 4 7 14 5 5 35 0 +
45 c Patient 9 82 53 49 36 60 0 +
46 Mia
Donor 6 35 35 29 7 40 0 +
47 Mia
Donor 4 29 16 19 10 9 0 +
48 Mia
Donor 3 23 13 14 8 12 0 +
49 Mia
Donor 3 17 13 9 2 12 0 +
50 Mia
Donor 2 10 5 2 2 3 0 +
51 Mia
Donor 5 39 6 12 11 14 0 +
52 Mia
Donor 5 39 14 17 7 7 0 +
53 Mia
Donor 6 50 19 25 9 11 0 +
54 Mia
Donor 3 34 9 9 5 3 0 +
55 Mia
Donor 3 37 7 14 3 5 0 +
56 Mia
Donor 3 19 5 6 3 5 0 +
57 Mia
Donor 8 71 28 38 14 12 0 +
58 Mia
Donor 5 32 14 8 5 17 0 +
59 Mia
Donor 7 46 9 12 5 19 0 +
60 Mia
Donor 5 23 5 9 2 17 0 +
61 Mia
Donor 3 20 3 3 5 3 0 +
62 Mia
Donor 5 29 5 6 5 11 0 +
63 Mia
Donor 3 33 9 11 10 3 0 +
64 Mia
Donor 5 25 17 18 12 10 0 +
65 Mia
Patient 2 38 8 6 5 5 0 +
66 Mia
Patient 3 39 6 10 3 9 0 +
67 Mia
Patient 2 14 4 4 2 1 0 +
68 Mia
Patient 2 35 10 10 2 4 0 +
69 Mia
Donor 3 21 9 14 2 15 0 +
70 Mia
Donor 2 16 17 14 4 10 0 +
71 Mia
Donor 3 35 16 19 6 7 0 +
72 Mia
Donor 2 14 4 4 4 2 0 +

No.
Anti From DAT
Negative
Control
C.C.C
73 Mia
Patient 2 7 2 2 4 4 0 +
74 Mia
Patient 2 14 4 4 2 4 0 +
75 Mia
Patient 2 17 3 6 3 2 0 +
76 Jka
Patient 3 14 4 5 2 12 0 +
77 Jka
Patient 5 36 29 26 23 40 0 +
78 Jka
Patient 4 31 29 23 20 22 0 +
79 Jka
Patient 5 32 27 26 13 30 0 +
80 Jka
Patient 2 27 12 9 2 18 0 +
81 Jka
Patient 4 28 30 23 19 30 0 +
82 Jka
Donor 5 17 16 18 7 24 0 +
83 Jka
Patient 2 30 10 11 4 18 0 +
84 Jka
Patient 2 29 8 9 4 17 0 +
85 Jka
Patient 7 48 27 28 21 32 0 +
86 Jkb
Donor 4 27 21 21 7 23 0 +
87 Jkb
Donor 7 54 32 27 18 29 0 +
88 Jkb
Patient 6 33 35 29 11 45 0 +
89 Jkb
Patient 5 25 28 28 20 36 0 +
90 Jkb
Patient 5 17 18 16 2 28 0 +
91 Jkb
Patient 7 39 22 16 9 21 0 +
92 Jkb
Patient 4 19 7 7 2 10 0 +
93 Jkb
Patient 2 9 5 4 3 9 0 +
94 Jkb
Patient 5 33 18 17 9 18 0 +
95 Jkb
Patient 5 20 15 19 8 19 0 +
96 Fya
Patient 6 64 57 58 36 38 0 +
97 Fya
Patient 5 54 27 24 10 17 0 +
98 Fya
Patient 3 41 12 7 3 7 0 +
99 Fya
Patient 4 50 27 27 3 3 0 +
100 Fya
Patient 3 20 16 11 6 9 0 +
101 Fya
Patient 4 27 19 11 4 9 0 +
102 Fya
Patient 5 51 51 45 35 41 0 +
103 Fyb
Patient 5 52 47 46 24 35 0 +
104 Fyb
Patient 3 20 18 20 18 20 0 +
105 Fyb
Patient 5 23 11 14 2 12 0 +
106 Fyb
Patient 5 42 28 16 10 16 0 +
107 Fyb
Patient 4 16 11 14 7 16 0 +
108 Dia
Patient 3 14 2 5 2 14 0 +

No.
Anti From DAT
Negative
Control
C.C.C
109 Dia
Patient 8 50 35 42 25 46 0 +
110 Dia
Patient 5 25 14 13 10 14 0 +
111 Dia
Patient 5 40 29 29 12 34 0 +
112 Dia
Patient 2 9 4 7 2 7 0 +
113 S Patient 6 46 27 30 11 22 0 +
114 E+c Patient 4 56 3 3 3 8 0 +
115 E+c Donor 12 82 57 47 49 65 0 +
116 E+c Patient 8 56 24 21 15 31 0 +
117 E+c Patient 6 39 16 13 2 22 0 +
118 E+c Patient 6 42 17 16 4 22 0 +
119 E+c Patient 2 12 4 4 2 4 0 +
120 E+c Patient 3 19 4 6 4 11 0 +
121 E+c Patient 3 10 2 2 2 9 0 +
122 E+c Patient 9 52 56 60 19 74 0 +
123 E+c Patient 3 32 18 16 7 22 0 +
124 C+e Patient 4 13 22 12 12 16 0 +
125 C+e Patient 9 52 70 73 29 81 0 +
126 C+e Patient 5 36 25 26 2 6 0 +
127 C+e Patient 2 11 6 7 6 9 0 +
128 C+e Patient 2 10 5 6 4 8 0 +
129 E+Jka
Patient 6 51 29 29 9 42 0 +
130 E+Jka
Patient 4 29 18 16 7 22 0 +
131 E+Jka
Patient 2 13 3 2 2 7 0 +
132 E+Jka
Patient 4 23 12 11 6 15 0 +
133 E+Jka
Patient 3 29 10 8 5 10 0 +
134 E+Jka
Patient 6 51 38 29 7 30 0 +
135 E+Jka
Patient 9 41 40 46 14 58 0 +
136 E+Jka
Patient 8 50 34 40 20 50 0 +
137 E+Jka
Patient 8 44 29 31 21 44 0 +
138 E+Mia
Patient 4 31 15 17 13 22 0 +
139 E+Mia
Patient 9 49 63 70 29 25 0 +
140 E+Mia
Patient 2 16 4 6 4 2 0 +
141 E+Mia
Patient 8 31 12 18 9 17 0 +
142 E+Mia
Patient 5 26 14 18 6 20 0 +
143 E+Mia
Patient 3 15 9 10 5 15 0 +
144 E+Mia
Patient 3 16 8 9 3 11 0 +

No.
Anti From DAT
Ab In
House
Kit Heat Ether
Negative
Control
C.C.C
145 E+Mia
Patient 3 16 8 10 4 10 0 +
146 S+Mia
Patient 5 35 9 10 7 12 0 +
147 S+Mia
Patient 5 38 14 22 13 20 0 +
148 S+Mia
Patient 2 15 6 4 4 8 0 +
149 S+Mia
Patient 4 20 10 15 9 13 0 +
150 S+Mia
Patient 5 29 14 15 14 20 0 +
151 Mia
+Jkb
Patient 3 21 6 7 7 8 0 +
152 S+Jka
Patient 4 26 12 12 10 13 0 +
153 E+c+Mia
Patient 7 42 29 26 12 37 0 +
154 E+c+Mia
Patient 7 48 30 22 12 36 0 +
155 E+c+Mia
Patient 5 33 25 20 10 31 0 +
156 E+c+Mia
Patient 3 25 7 7 5 12 0 +
157 E+c+Mia
Patient 5 30 20 21 4 12 0 +
158 E+c+Mia
Patient 4 37 16 12 10 25 0 +
159 E+c+Mia
Patient 4 18 22 23 16 22 0 +
160 E+c+Mia
Patient 6 52 28 36 13 31 0 +
161 E+c+Mia
Patient 4 43 16 16 5 4 0 +
162 E+c+Mia
Patient 5 38 17 16 9 25 0 +
163 E+c+Mia
Patient 6 56 47 49 20 24 0 +
164 E+c+Jka
Patient 7 57 20 18 13 36 0 +
165 E+c+Jka
Patient 4 19 11 7 4 12 0 +
166 E+c+Jka
Patient 5 41 30 23 9 16 0 +
167 E+c+Jka
Patient 10 70 52 40 27 59 0 +
168 E+c+Jka
Patient 4 63 31 33 11 51 0 +
169 E+c+Jka
Patient 2 21 6 7 2 11 0 +
170 E+c+Jka
Patient 3 22 10 8 3 17 0 +
171 E+c+Jka
Patient 6 42 22 26 13 21 0 +
172 E+c+Jka
Patient 3 28 16 17 2 17 0 +
173 E+c+Jka
Patient 8 52 37 35 13 45 0 +
174 E+S+Dia
Patient 4 25 20 27 18 21 0 +
175 E+Mia
+Jka
Patient 9 72 37 38 26 49 0 +

Explanation of Table 4
1. Column Anti is antibodies that use for sensitization.
2. Column From is antibodies that received from donors and patients.
3. Column DAT is scores of direct antiglobulin positive red blood cells after
sensitization.
4. Column Ab is sum scores of antibodies titration before sensitization.
5. Column In house is sum scores of antibodies titration of In house Acid/EDTA
Elution method.
6. Column Kit is sum scores of antibodies titration of DiaCidel Elution Kit
method.
7. Column Heat is sum scores of antibodies titration of Heat Elution method.
8. Column Ether is sum scores of antibodies titration of Ether Elution method.
9. Column Negative Control is parallel test of last wash supernatant after
sensitized.
10. Column C.C.C is test of Coombs Control Cell for confirm Negative Control is
real negative.
3. Statistical Analysis
As shown in Table 5-21, Two-sample paired t-test used by SPSS version 13.0 to
calculate the titration mean scores, mean differences, standard deviations (SD) and
significant (2-tailed) / to comparing the results, the mean scores of antibody titration
those showed the efficiency potential of two elution methods which were statistically
significant if P 0.05.
Table 5. Comparison of Mean Scores among In house, Kit, Heat and Ether for
Anti-D
Anti-D (n=20) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)
Pair 1 (In house-Kit) 28.35-26.40 1.950 4.430 .064
Pair 2 (In house-Heat) 28.35-14.30 14.050 8.062 .000
Pair 3 (In house-Ether) 28.35-37.15 -8.800 5.736 .000
Pair 4 (Kit-Heat) 26.40-14.30 12.100 9.130 .000
Pair 5 (Kit-Ether) 26.40-37.15 -10.750 6.843 .000
Pair 6 (Heat-Ether) 14.30-37.15 -22.850 11.151 .000

For Anti-D, the mean scores in following order: Ether > In house kit > DiaCidel
kit > Heat. However, Pair 1 (In house-Kit) is not significant (p > 0.05).
Anti-E
Anti-E (n=21) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)
Pair 1 (In house-Kit) 13.48-11.95 1.524 5.492 .218
Pair 4 (Kit-Heat) 11.95-8.14 3.810 8.524 .054
Pair 5 (Kit-Ether) 11.95-24.95 -13.000 8.832 .000
Pair 6 (Heat-Ether) 8.14-24.95 -16.810 9.474 .000
For Anti-E, the mean scores in following order: Ether > In house kit > DiaCidel
kit > Heat. Pair 1 (In house-Kit) and Pair 4 (Kit-Heat) are not significant (p > 0.05).
Anti-Mia
Anti-Mia
(n=30) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)
Pair 1 (In house-Kit) 10.53-11.80 -1.267 3.723 .073
Pair 3 (In house-Ether) 10.53-9.20 1.333 6.200 .248
Pair 4 (Kit-Heat) 11.80-5.40 6.400 6.425 .000
Pair 5 (Kit-Ether) 11.80-9.20 2.600 7.668 .073
Pair 6 (Heat-Ether) 5.40-9.20 -3.800 7.694 .011
For Anti-Mia
, the mean scores in following order: DiaCidel kit > In house kit >
Ether > Heat. Pair 1 (In house-Kit), Pair 3 (In house-Ether), Pair 5 (Kit-Ether) and
Pair 6 (Heat-Ether) are not significant (p > 0.05).

Anti-Jka
Anti-Jka
Pair 1 (In house-Kit) 19.20-17.80 1.400 3.204 .200
Pair 4 (Kit-Heat) 17.80-11.50 6.300 3.466 .000
Pair 5 (Kit-Ether) 17.80-24.30 -6.500 3.866 .000
Pair 6 (Heat-Ether) 11.50-24.30 -12.800 4.662 .000
For Anti-Jka
, the mean scores in following order: Ether > In house kit >
DiaCidel kit > Heat. Pair 1 (In house-Kit) and Pair 3 (In house-Ether) are not
significant (p > 0.05).
Anti-Jkb
Anti-Jkb
Pair 1 (In house-Kit) 20.10-18.40 1.700 3.164 .124
Pair 4 (Kit-Heat) 18.40-8.90 9.500 4.927 .000
Pair 5 (Kit-Ether) 18.40-23.80 -5.400 5.168 .009
Pair 6 (Heat-Ether) 8.90-23.80 -14.900 8.762 .000
For Anti- Jkb
DiaCidel kit > Heat. Pair 1 (In house-Kit), Pair 3 (In house-Ether) and Pair 5 (Kit-
Ether) are not significant (p > 0.05).

Anti-Fya
Anti-Fya
Pair 1 (In house-Kit) 29.86-26.14 3.714 3.251 .023
Pair 4 (Kit-Heat) 26.14-13.86 12.286 8.056 .007
Pair 5 (Kit-Ether) 26.14-17.71 8.429 9.589 .059
Pair 6 (Heat-Ether) 13.86-17.71 -3.857 2.410 .005
For Anti- Fya
, the mean scores in following order: In house kit > DiaCidel kit >
Ether > Heat. Pair 1 (In house-Kit), Pair 4 (Kit-Heat) and Pair 5 (Kit-Ether) are not
Anti-Fyb
Anti- Fyb
Pair 1 (In house-Kit) 23.00-22.00 1.000 6.364 .743
Pair 4 (Kit-Heat) 22.00-12.20 9.800 7.694 .046
Pair 5 (Kit-Ether) 22.00-19.80 2.200 5.119 .391
Pair 6 (Heat-Ether) 12.20-19.80 -7.600 3.647 .010
For Anti- Fyb
, the mean scores in following order: In house kit > DiaCidel kit >
Ether > Heat. All pairs are not significant (p > 0.05).
Anti-Dia
Anti- Dia
Pair 1 (In house-Kit) 16.80-19.20 -2.400 3.130 .162
Pair 4 (Kit-Heat) 19.20-10.20 9.000 7.348 .052
Pair 5 (Kit-Ether) 19.20-23.00 -3.800 3.564 .076
Pair 6 (Heat-Ether) 10.20-23.00 -12.800 8.526 .028

For Anti-Dia
, the mean scores in following order: Ether > DiaCidel kit > In
house kit > Heat. All pairs are not significant (p > 0.05).
Anti-E+c
Anti- E+c (n=10) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)
Pair 1 (In house-Kit) 20.10-18.80 1.300 3.743 .301
Pair 4 (Kit-Heat) 18.80-10.70 8.100 12.556 .072
Pair 5 (Kit-Ether) 18.80-26.80 -8.000 5.077 .001
Pair 6 (Heat-Ether) 10.70-26.80 -16.100 14.985 .008
For Anti- E+c, the mean scores in following order: Ether > In house kit >
DiaCidel kit > Heat. Pair 1 (In house-Kit), Pair 2 (In house-Heat), Pair 4 (Kit-Heat)
and Pair 6 (Heat-Ether) are not significant (p > 0.05).
Anti-C+e
Anti- C+e (n=5) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)
Pair 1 (In house-Kit) 25.60-24.80 .800 5.215 .749
Pair 4 (Kit-Heat) 24.80-10.60 14.200 19.422 .177
Pair 5 (Kit-Ether) 24.80-24.00 .800 11.009 .879
Pair 6 (Heat-Ether) 10.60-24.00 -13.400 21.582 .237
For Anti-C+e, the mean scores in following order: Ether > In house kit >
DiaCidel kit > Heat. All pairs are not significant (p > 0.05).

Anti-E+Mia
Anti- E+Mia
Pair 1 (In house-Kit) 16.63-19.75 -3.125 2.295 .006
Pair 4 (Kit-Heat) 19.75-9.13 10.625 12.648 .049
Pair 5 (Kit-Ether) 19.75-15.25 4.500 16.639 .469
Pair 6 (Heat-Ether) 9.13-15.25 -6.125 6.105 .025
For Anti- E+Mia
, the mean scores in following order: DiaCidel kit > In house kit
> Ether > Heat. All pairs are not significant (p > 0.05).
Anti-E+Jka
Anti- E+Jka
Pair 1 (In house-Kit) 23.67-23.56 .111 4.567 .944
Pair 4 (Kit-Heat) 23.56-10.11 13.444 10.584 .005
Pair 5 (Kit-Ether) 23.56-30.89 -7.333 4.743 .002
Pair 6 (Heat-Ether) 10.11-30.89 -20.778 13.479 .002
For Anti- E+Jka
DiaCidel kit > Heat. Pair 1 (In house-Kit) and Pair 3 (In house-Ether) are not
Anti-S+Mia
Anti- S+Mia
Pair 1 (In house-Kit) 10.60-13.20 -2.600 3.912 .211
Pair 2 (In house-Heat) 10.60-9.40 1.200 .837 .033
Pair 4 (Kit-Heat) 13.20-9.40 3.800 3.701 .083
Pair 5 (Kit-Ether) 13.20-14.60 -1.400 3.286 .395
Pair 6 (Heat-Ether) 9.40-14.60 -5.200 1.304 .001

For Anti- S+Mia
, the mean scores in following order: Ether > DiaCidel kit > In
house kit > Heat. All pairs are not significant (p > 0.05) except Pair 6 (Heat-Ether) (p
< 0.05).
Anti-E+c+Mia
Anti- E+c+Mia
Pair 1 (In house-Kit) 23.36-22.55 .818 4.215 .534
Pair 3 (In house-Ether) 23.36-23.55 -.182 10.255 .954
Pair 4 (Kit-Heat) 22.55-10.55 12.000 8.355 .001
Pair 5 (Kit-Ether) 22.55-23.55 -1.000 12.562 .797
Pair 6 (Heat-Ether) 10.55-23.55 -13.000 8.683 .001
For Anti- E+c+Mia
Anti-E+c+Jka
Anti- E+c+Jka
Pair 1 (In house-Kit) 23.50-21.40 2.100 4.701 .191
Pair 4 (Kit-Heat) 21.40-9.70 11.700 7.009 .001
Pair 5 (Kit-Ether) 21.40-28.50 -7.100 9.433 .041
Pair 6 (Heat-Ether) 9.70-28.50 -18.800 12.118 .001
For Anti- E+c+Jka

Combined two Ab
Combined two Ab
(n=39)
Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)
Pair 1 (In house-Kit) 19.18-19.67 -.487 4.192 .472
Pair 4 (Kit-Heat) 19.67-9.95 9.718 12.124 .000
Pair 5 (Kit-Ether) 19.67-22.62 -2.949 10.052 .075
Pair 6 (Heat-Ether) 9.95-22.62 -12.667 13.735 .000
For Combined two Ab, the mean scores in following order: Ether > DiaCidel kit
> In house kit > Heat. Pair 1 (In house-Kit), Pair 3 (In house-Ether) and Pair 5 (Kit-
Combined three Ab
Combined three Ab
(n=23)
Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)
Pair 1 (In house-Kit) 23.87-22.91 .957 4.557 .325
Pair 4 (Kit-Heat) 22.91-11.17 11.739 7.225 .000
Pair 5 (Kit-Ether) 22.91-26.70 -3.783 11.123 .117
Pair 6 (Heat-Ether) 11.17-26.70 -15.522 10.587 .000
For Combined three Ab, the mean scores in following order: Ether > In house kit
> DiaCidel kit > Heat. Pair 1 (In house-Kit), Pair 3 (In house-Ether) and Pair 5 (Kit-
Outputs from SPSS version 13.0 of all antibodies are showed in Appendix.

CHAPTER VI
DISCUSSION
The purpose of this study was to compare the efficiency of four elution methods.
DiaCidel elution kit was the first method that was interesting but very expensive. So
the attempt to develop In house Acid/EDTA was occurred because the chemical
reagents used in the preparation of Acid/EDTA are the common chemical reagents in
blood blank. In this study, 175 samples of antibodies collected from blood donors and
patients were divided into three groups; single antibodies, combined two antibodies
and combined three antibodies. All were clinically significant antibodies that always
occurred in Thai people. The red blood cells tested for antibodies sensitization were
groups O with known antigenic profile and tested with direct antiglobulin negative.
In house Acid/EDTA is different from DiaCidel elution kit in terms of method of
preparation, using and storage. The preparation of In house kit is referred to P.C.
Byrne (25) that composed of 4 reagents; 10 EDTA, 0.1M glycine-HCl buffer (pH
1.5), 1.0M TRIS-NaCl and normal saline used as washing solution. DiaCidel elution
kit contains 3 reagents; Washing solution, Elution solution and Buffer solution. The
Kit is easier to use than In house Acid/EDTA because Elution solution contain
Methylene blue that give changing form yellow to blue which indicated the pH from
acid to base. Elution solution of In house Acid/EDTA is the mixture of 10 EDTA
and 0.1M glycine-HCl buffer (pH 1.5) and buffer solution is 1.0M TRIS-NaCl.
Because the In house has no pH indicator, the pH papers were to be used to adjust pH
which is manually difficult.
However, disadvantage of the commercial Elution Kits was demonstrated by
Leger R.M. et al. Commercial washing solution used for acid elution are low-ionic-
strength and normally yielded higher eluates, but in cases of high-titer antibodies, the
results of eluates can be false-positive. They belief that the low-ionic-strength of wash
solution caused aggregation of IgG and nonspecific attachment of IgG on RBCs.
Aggregation will contain IgG serum antibodies in proportion to the titer of the
antibody that is eluted from antigen-negative RBCs.(29)

Kallaya Kirdkoungam Discussion/40
This study used SPSS version 13.0 to calculate mean scores of antibodies
titration and compare mean by paired t-test. Calculation was separated by type of
antibodies because each type of antibodies is different in structures and reactions.
There are 15 types of antibodies; the single antibodies are Anti-D, Anti-E, Anti-Mia
,
Anti-Jka
, Anti-Jkb
, Anti-Fya
, Anti-Fyb
and Anti-Dia
; the combined antibodies are Anti-
E+c, Anti-C+e, Anti-E+ Mia
, Anti-E+ Jka
, Anti-S+ Mia
, Anti-E+c+ Mia
and Anti-
E+c+Jka
.
Rh system antibody is one of the most important antibodies. These antibodies
are human immune response that causes HTR and HDN. They are always found in
donors and patients (30), in this experiment, most samples chosen were in Rh system.
For Anti-D, the results showed that Ether method was better than other methods
because it gave highest mean scores. Both In house Acid/EDTA and DiaCidel kit
were not different in elution potential (p = .064). Heat 56 ºC is the worst method for
this antibody. For Anti-E, the result was similar to Anti-D, Ether was better than other
methods. In house Acid/EDTA and DiaCidel kit were not significantly different in
their potential (p = .218). Heat 56 ºC was the worst method. For combined Rh
antibodies, Anti-E+c, In house Acid/EDTA and DiaCidel kit were not different in
their potential (p = .301) but they gave low potential than Ether and Heat 56 ºC gave
the lowest potential. Anti-C+e, the results could not be interpreted because every pair
showed no statistical significant. In conclusion of Rh system Ether was the better than
other method and Heat was the worst method. In house and Kit are not different in
their potency.
The combination of Rh antibodies to other systems such as Anti-E+ Mia
, Anti-
E+ Jka
, Anti-E+c+ Mia
and Anti-E+c+Jka
gave results dissimilarly to single and
combined Rh antibodies. For Anti-E+ Mia
, DiaCidel Kit was the best method and high
potency than In house (p = .006) but it was not different form Ether (p =.469). Heat
was the lowest potency for this antibody. For Anti-E+Jka
, Ether method was highest
potency method. In house and DiaCidel Kit were not different in their elution potency
(p = .944). Heat was lowest potency methods. The results of the three combined of
antibodies Anti-E+c+ Mia
, Ether was the highest potency method. DiaCidel Kit and In
house were not different in their elution potency (p = .534). Heat method was the
lowest potency. The result of Anti-E+c+Jka
was similar to Anti-E+c+ Mia
. Ether was

the highest potency method. DiaCidel Kit and In house were not different in their
elution potency (p = .191). Heat method was the lowest potency.
For overview of Rh system antibodies, it was shown that Ether method was the
best method for elution because the eluates gave the maximum means scores in all
antibodies. The exception is Anti-Rh plus Anti-Mia
which the results were not
different; eluates mean score of both Acid/EDTA methods were equivalent to Ether
method in elution potential. In house method and Kit showed that they were not
different in capability. Heat 56 °C has lowest capable in elution for this system. Our
results in Rh system were parallel to the research of Nathalang O. et al. but their
study did not have variety of antibodies. Most samples were single Rh antibody such
as Anti-D, Anti-C, Anti-E, Anti-c and anti-e and had only one mixture antibody that
was Anti-C+e. (27)
In MNSs system, Anti-Mia
and Anti-S were chosen because most of them are
IgG antibodies while of M and N are mostly IgM. (31) Anti-Mia
which is natural
occurring or immunizing is always found in both Thai patients and donors. The
incidence of Mia
antigen is 9.72% in the Thai population which is higher than that
found in other populations, and so far, as known, is the highest in the worlds (32) so it
is one of the most important antibodies in blood transfusion in Thailand. Single Anti-
S is very rare, there is only one sample (sample No. 113) in this experiment but there
are 5 samples of the combination of Anti-S and Anti-Mia
. For Anti-Mia
the result
showed that In house Acid/EDTA was not different from DiaCidel kit in their potency
(p = .073). Ether gave lower mean scores than both Acid/EDTA methods but was not
significantly different in their potency (p =.248, .073) Heat method gave the lowest
potential. For Anti-S+Mia
, In house was similar to DiaCidel kit in their potency (p =
.211). In house Acid/EDTA gave lower potency than Ether (p = .009) and DiaCidel
kit methods were not better than Ether (p =.395) for MNSs system antibodies. For
Anti-Mia
, our results were similar to the research of Nathalang O. et al. which
Acid/EDTA gave highest mean scores and Ether gave lowest mean scores. (27)
Kidd antibodies are clinically significant since they can cause acute and delayed
transfusion reactions. (13) Two antigens, Jka
and Jkb
, there are the three common
phenotypes Jka-b+
, Jka+b-
and Jka+b+
. Both antigens can be found in normal Asians. (11)
The very rare phenotype is Jka-b-
, but can be found in Asians and Polynesians. (11)

Samples which were selected in this study had 10 Anti-Jka
and 10 Anti-Jkb
. For Anti-
Jka
, Ether method gave the highest potency. In house Acid/EDTA and DiaCidel kit
showed no difference in their potency (p = .200). In the same way, Anti-Jkb
gave the
result of In house Acid/EDTA and DiaCidel kit no different in their potency (p =
.124). Ether method gave the highest potency and Heat method gave the lowest
potency for single antibodies in this system. For Nathalang O. et al. research, the
result of Anti-Jka
was Acid/EDTA gave higher mean scores than Ether and Anti-Jkb
was Ether gave higher mean scores than Acid/EDTA. Heat gave the lowest mean
scores for this system. (27)
For antibodies in Duffy system, single Anti-Fya
and single Anti-Fyb
, are much
immunized antibodies in Thai patients. They always combine with other antibodies.
(30) In this research there were 7 single Anti-Fya
, 5 single Anti-Fyb
samples and the
combination of Anti-Fya
or Anti-Fyb
with others were not. For Anti-Fya
, In house
Acid/EDTA and DiaCidel Kit were not different in their potency (p= .023). In house
was the best method for this antibody. For Anti-Fyb
, In house Acid/EDTA (p= .430)
and DiaCidel Kit (p= .391) were better than Ether. In house Acid/EDTA and DiaCidel
Kit were not different in their potency (p= .743). The results of two kinds of
antibodies in Duffy system are not different (n= 5).
The role of anti-Dia
antibody in causing hemolytic disease of newborn was first
recognized in 1955. Genetic studies reveal that there is great variation in the
distribution of the Dia
antigen in different populations. It is very rare among
Caucasians, but is relatively common (5%-15%) among South American Indians and
Asia populations. (33) For Anti-Dia
, the results were not different in every method.
The summery in this system can not interpretation. The finding of more samples is
necessary.
The outline calculation of whole antibodies made by separated four big groups
of antibodies such as combined two and combined three antibodies and total
antibodies. The calculation result of combined two antibodies In house and Kit were
equal in their elution effectiveness that they were no statistical significance (p = .472).
Ether was the highest potency for this antibody. For combined three antibodies, mean
scores showed that Ether potential was better than both Acid/EDTA and Heat. In

house was the higher potency than Kit, Heat and Ether. This was demonstrate that In
house was the best method if consider in whole antibodies.
The summarization of all antibodies in this study indicate that Ether gave the
highest yield antibodies if consider separated by type of antibodies. Vos and Kelsall
proposed that organic solvents, such as ether, denature or destroy antigens, whereas
antibody molecules are not affected. Perhaps his might occur by dissolution of the
RBC membrane bilipid layer.(4) For Rh system that Ether method gave the highest
yield mean scores, Hughes-Jones NC et al. suggested that ether acts by altering the
tertiary structure of antibodies. Either would obviously disturb structural
complementarities. (4) However, hazard to use ether must be considered. Ether is
dangerous for blood banker. Although Fume Hood that has filters for absorb organic
solvent is one way that was use these method safely but it was not available in our
laboratory and the filters is very expensive. The Hood that was used in this research
has not filter which ether remain and released to the environment that is not good for
our global. Acid/EDTA is the safer method than Ether although in some types of
antibodies such as Anti-Rh it has lower capability than Ether. Although Ether gave
high yield eluates antibodies, but its disadvantage is that the red blood cells are
destroyed by hemolytic. The harvesting of red blood cells after elutes for phenotype
minor blood group are impossible and eluates are contaminated with hemoglobin
which made deep red so that eluates are difficult to be used for antibody identification
especially IgM antibodies which do not have wash step. In the opposite way, both
method of Acid/EDTA do not destroy red blood cells which are remained after elutes
and were capable for phenotyping. The remaining red blood cells from In house
Acid/EDTA are not hemolytic and are not different from normal red blood cells.
However, DiaCidel Elution Kit gave the remaining red blood cells after elutes in bad
appearance similar to clot blood which phenotyping may be difficult.
There are few studies that compared methods of elution. Most researches were
tested for one elution method. However, there are four researches that compared
several methods. The first is research of Nathalang O. et al. that there are nine
antibodies: Anti-D, Anti-C, Anti-C+e, Anti-c, Anti-E, Anti-e, Anti-Jka
, Anti-Jkb
and
Anti-Mia
. They compared result three elution methods including heat, ether and
DiaCidel elution kit (acid EDTA). The second is research of Rekvig OP. and

Hannestad K., they selected Human IgM and IgG antibodies such as Rh, Kell, Duffy,
ABO system against blood group antigens is A, B, D, C, c, E, e, Fya
, K, auto
antibodies and mouse IgM and IgG antibodies against sheep erythrocytes have been
eluted from intact human and sheep red cells by glycine-HCl buffer (pH 3.0). The
yield of human was higher with acid than with heat and ether elution, and the
contamination of hemoglobin in the eluate was negligible. (5) The third is research of
S.F. South et al. which compared eleven elution methods for their efficacy in
recovering antibodies from red blood cells sensitized with immunoglobulin.
Antibodies that they selected were Rh, Kell, MNSs, Duffy, Kidd, ABO system, Anti-
Kpb
, Anti-Vel and Anti-Ge. Methods that they selected were Lui, heat, digitonin-acid,
ether, chloroform, dichloromethane, xylene, and alcohol freeze-thaw and three
commercial elution kits were EluAid, Ortho Diagnostic System Inc.; Elution Solution,
Biological Corporation of American; and Elu-Kit II, Gamma Biological, Inc. Their
results were the xylene elution method proved to be the most effective method,
followed by Elu-Kit II, chloroform, dichloromethane, and digitonin-acid. The other
six methods evaluated were not optimal based on the suitability of each methods and
the calculated sensitivity. (6) The fourth is research of Burin des Roziers N. and
Squalli S. In their study, they compared the relative abilities of chloroquine
diphosphate dissociation, acid/EDTA elution, and heating at 56 ºc for 10 minutes to
generate intact antibody-free RBCs from 50 DAT-positive RBCs coated in vitro or in
vivo, and then they assessed the integrity of common blood group antigens. The
following all antibodies were studies: Anti-D, Anti-C, Anti-c, Anti-E, Anti-e, Anti-K,
Anti-k, Anti-Kpa
, Anti-Kpb
, Anti-Fya
, Anti-Fyb
, AntiJka
, Anti-S and Anti-s. In
addition, the activities of the antibodies eluted by the acid/EDTA method or with heat
were compared. Their results were the agglutination scores of acid/EDTA eluates
were higher than those obtained after heat elution in 43 of 50 samples (p<0.0001) and
similar in 3 samples. In only 4 samples (1 anti-S, 1 anti-s, and 2 anti-E) heat elution
was more effective than acid/EDTA. (43) The results of four researches that above-
mentioned are similar to our results which organic solvents elution method and
acid/EDTA elution method are superior heat 56 ºc elution method in their efficiency
of recovering antibodies from red blood cells.

For this research, there are 175 samples antibodies composed of single,
combined two and combined three antibodies. They are clinically significant and
covered every blood group systems those important in blood transfusion. The total of
sample size and types of antibodies in this experiment are much more than the other
four researches mentioned above. The best elution method is the method that can be
eluting every antibody sensitized on patient’s red blood cells especially in case that
there are combined antibodies in serum. In case of HDN, it is very important to detect
and identify absolutely every antibody those sensitized on patients red blood cells and
choose the blood free from those antigens for the most transfusion safety. So there are
more than one method of elution in reference laboratory help complete elutes
antibodies from patients red blood cells because there is no method that good for
every antibodies example heat elution is the best method for IgM but it is not good for
IgG but other method are good for it. In present, elution method that can keep red
blood cells for phenotyping is the most interesting because HDN babies blood
samples are low volume and blood collection several times are not safe for new born
babies which low whole blood in circulation.
The comparison of price between DiaCidel Kit Elution and In house Acid/EDTA
showed that the kit is very expensive. The price is two hundred baths per test, while In
house Acid/EDTA is very cheap; one bath per test. On this study, it was shown that
both Acid/EDTD methods were not different in their potency for all types of
antibodies elution. Therefore, In house kit is still in developing for convenient use
which will give a new selective way for elution with high efficiency and very cheap.
The preparation of In house Acid/EDTA in one time can prepare chemical more than
one hundred tests. Heat is old method that gives the lowest yield in every type of IgG
antibodies but it is good for IgM antibodies especially in ABO system. (4) The price
of Heat method was 1.28 baths per test; it is more expensive than In house and Ether
methods but cheaper than commercial kit. The price of Ether was 0.45 baths per test
so Ether was the cheapest method and it gave very high effectiveness.

Kallaya Kirdkoungam Conclusion/46
CHAPTER VII
CONCLUSION
The comparisons of four methods for elution such as In House Acid/EDTA,
DiaCidel Elution Kit, Heat and Ether showed that Ether is the method that gave
highest potency in separated type of antibodies, especially antibodies in Rh system,
however, it is very hazard so it is not the best way that is selected to use in the
laboratory. DiaCidel Elution Kit and In House Acid/EDTA are safer and gave high
potency in every type of antibodies, although they give lower yield than Ether but
they are better than old Heat method. In House is the best method because it is
equivalent to DiaCidel Elution Kit in effectiveness of elution but the price is lower
than Kit and it gave highest elution potential in whole antibodies. Nevertheless the
improvement of In House Acid/EDTA is continuing. For Heat method, although it is
not good for IgG antibodies but most researches showed that it is high effectiveness
for IgM antibodies especially in ABO system.

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APPENDIX

Kallaya Kirdkoungam Appendix /52
APPENDIX
Cost of In House Acid-glycine/EDTA
1. Ten percent EDTA (disodium ethyleneduamine-tetraacetate)
EDTA 100 g = 3,230 bath
EDTA 2 g = (3,230x2)/ 100 = 64.6 bath
Distilled water 1,000 ml = 7 bath
Distilled water 20 ml = 0.14 bath
Make up to 10% EDTA 20 ml = 64.6+0.14 = 64.74 bath
Using 250 l/test = [64.74x250]/20x1000 = 0.80925 bath
2. Zero point one M glycine-HCl buffer (pH 1.5)
Glycine 500 g = 2,130 bath
Glycine 0.75 g = (2,130x0.75)/500 = 3.195 bath
HCl 2.5 L = 428 bath
HCl 2 ml = (428x2)/2.5x1000 = 0.3424 bath
Zero point nine percent Normal Saline 500 ml = 20 bath
Zero point nine percent Normal Saline 100 ml = (20x100)/500 = 4 bath
Glycine-HCl buffer 100 ml = 3.195+0.3424+ 4 = 7.5347 bath
Using 1 ml/test = 7.5347 /100 = 0.075374 bath
3. One M TRIS-NaCl
TRIS (Tris(hydroxymethyl)aminomethan) 1,000 g = 4,880 bath
TRIS (Tris(hydroxymethyl)aminomethan) 12.1 g = (12.1x4,880)/1,000 =
59.048 bath
Sodium Chloride 1,000 g = 2,810 bath
Sodium Chloride 5.25g = (5.25x2, 810)/1,000 = 14.7525 bath
Distilled water 1,000 ml = 7 bath
Distilled water 100 ml = 0.7 bath
Make up to 1.0 M TRIS-NaCl 100 ml = 59.048+14.7525+0.7 = 74.5005
bath
Using 130 l/test=[74.5005x130]/100x1000 =0.09685065 bath

Total = 0.80925+0.075374+0.09685065 = 0.98147465 bath
Cost of in house acid-glycine/EDTA 1 bath/test
Cost of Heat 56 °C
6% Bovine Albumin 1 ml/test = 1.28 bath
Cost of Heat-Ether 37 °C
Diethyl ether 1 ml/test = 0.45 bath

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