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 Kp

b 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 Jk

b, 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-Jk

b

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-Fy

a/ 5 anti-Fy

b/ 10 anti-Jk

a/

10 anti-Jkb/1 anti-S/ 8 anti-E+ Mi

a/ 9 anti-E+ Jk

a/ 5 anti-S+Mi

a/10 anti-E+c/ 5 anti-

C+e/ 5 anti-Dia/ 11/anti-E+c+Mi

a/ 10 anti-E+c+Jk

a/1 anti-E+S+Di

a/1 anti-Mi

a+Jk

b/ 1

anti-E+Mia+Jk

a/ and 1 anti-S+Jk

a.

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 Dilution Sample No.

n 2 4 8 16 32 64 128 256 512 Sum

Grade 3+ 3+ 3+ 2+ 2+ 2+ 1+ + + 0 none Sample 1

Score 10 10 10 8 8 8 5 3 2 0 64

Grade 4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+ + 0 none Sample 2

Score 12 12 12 10 10 8 8 5 3 0 80

Grade 1+ 1+ 1+ 1+ + + + + + 0 none Sample 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 Titration Sample

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 (n=10) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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 (n=10) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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, 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-

Ether) are not significant (p > 0.05).




Anti-Fya

Anti-Fya (n=7) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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 (n=5) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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 (n=5) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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 (n=8) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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 (n=9) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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, 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



Anti-S+Mia

Anti- S+Mia (n=5) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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 (n=11) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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, the mean scores in following order: Ether > In house kit >




Anti-E+c+Jka

Anti- E+c+Jka (n=10) Mean Mean difference Std. Deviation (SD) Sig. (2-tailed)

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, the mean scores in following order: Ether > In house kit >






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-Jk

b, Anti-Fy

a, Anti-Fy

b and Anti-Di

a; the combined antibodies are Anti-

E+c, Anti-C+e, Anti-E+ Mia, Anti-E+ Jk

a, Anti-S+ Mi

a, Anti-E+c+ Mi

a 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+ Mi

a and Anti-E+c+Jk

a 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+ Mi

a. 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-Mi

a 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-Jk

b. 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-Jk

b

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-Fy

b, 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-Fy

b samples and the

combination of Anti-Fya or Anti-Fy

b with others were not. For Anti-Fy

a, 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-Jk

b 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-Kp

b, Anti-Fy

a, Anti-Fy

b, AntiJk

a, 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



Example for complete antigenic profile red blood cell

Rh MNSs P Lewis Kidd Duffy Kell Diego Rh

D C E c e M N S s P1 Lea Leb

Mia

Jka Jkb Fya Fyb

K k Dia Dib

R1R1 + + 0 0 + + + 0 + + + 0 0 + + + 0 0 + 0 +



Mia

Jka Jkb Fya Fyb

K k Dia Dib

R1R2 + + + + + 0 + + + 0 0 + + + + + 0 0 + + +



Mia

Jka Jkb Fya Fyb

K k Dia Dib

R1r + + 0 + + + + 0 + + 0 0 + 0 + + + + + 0 +



Mia

Jka Jkb Fya Fyb

K k Dia Dib

R1Rz + + + 0 + + + + + + 0 + 0 + 0 + 0 0 + 0 +



Mia

Jka Jkb Fya Fyb

K k Dia Dib

R2R2 + 0 + + 0 + + 0 + + + 0 + + + + + + + 0 +



Anti-D

T-Test�

Paired Samples Statistics

Mean Std. Deviation

Std. Error

Mean

SCORE1 28.35 20 12.832 2.869 Pair 1

SCORE2 26.40 20 12.054 2.695

SCORE1 28.35 20 12.832 2.869 Pair 2

SCORE3 14.30 20 8.682 1.941

SCORE1 28.35 20 12.832 2.869 Pair 3

SCORE4 37.15 20 14.702 3.287

SCORE2 26.40 20 12.054 2.695 Pair 4

SCORE3 14.30 20 8.682 1.941

SCORE2 26.40 20 12.054 2.695 Pair 5

SCORE4 37.15 20 14.702 3.287

SCORE3 14.30 20 8.682 1.941 Pair 6

SCORE4 37.15 20 14.702 3.287

Paired Samples Correlations

N Correlation Sig.

Pair 1 SCORE1 & SCORE2 20 .939 .000








Anti-D (Continued)

Paired Samples Test

Paired Differences

95% Confidence

Interval of the

Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 1.950 4.430 .991 -.124 4.024

Pair 2 SCORE1 - SCORE3 14.050 8.062 1.803 10.277 17.823

Pair 3 SCORE1 - SCORE4 -8.800 5.736 1.283 -11.485 -6.115

Pair 4 SCORE2 - SCORE3 12.100 9.130 2.042 7.827 16.373

Pair 5 SCORE2 - SCORE4 -10.750 6.843 1.530 -13.953 -7.547

Pair 6 SCORE3 - SCORE4 -22.850 11.151 2.493 -28.069 -17.631

Paired Samples Test

t df Sig. (2-tailed)

Pair 1 SCORE1 - SCORE2 1.968 19 .064

Pair 2 SCORE1 - SCORE3 7.794 19 .000

Pair 3 SCORE1 - SCORE4 -6.861 19 .000

Pair 4 SCORE2 - SCORE3 5.927 19 .000

Pair 5 SCORE2 - SCORE4 -7.025 19 .000

Pair 6 SCORE3 - SCORE4 -9.164 19 .000

SCORE1 = In house Acid/EDTA

SCORE2 = DiaCidel Elution Kit

SCORE3 = Heat 56 ºC

SCORE4 = Heat Ether 37 ºC



Anti-E

T-Test


Mean N Std. Deviation

Std. Error

Mean

SCORE1 13.48 21 9.059 1.977 Pair 1

SCORE2 11.95 21 8.352 1.822

SCORE1 13.48 21 9.059 1.977 Pair 2

SCORE3 8.14 21 8.113 1.771

SCORE1 13.48 21 9.059 1.977 Pair 3

SCORE4 24.95 21 12.886 2.812

SCORE2 11.95 21 8.352 1.822 Pair 4

SCORE3 8.14 21 8.113 1.771

SCORE2 11.95 21 8.352 1.822 Pair 5

SCORE4 24.95 21 12.886 2.812

SCORE3 8.14 21 8.113 1.771 Pair 6

SCORE4 24.95 21 12.886 2.812


N Correlation Sig.









Anti-E (Continued)

Paired Samples Test

Paired Differences

95% Confidence

Interval of the

Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 1.524 5.492 1.198 -.976 4.024

Pair 2 SCORE1 - SCORE3 5.333 7.445 1.625 1.944 8.722

Pair 3 SCORE1 - SCORE4 -11.476 6.630 1.447 -14.494 -8.458

Pair 4 SCORE2 - SCORE3 3.810 8.524 1.860 -.071 7.690

Pair 5 SCORE2 - SCORE4 -13.000 8.832 1.927 -17.020 -8.980

Pair 6 SCORE3 - SCORE4 -16.810 9.474 2.067 -21.122 -12.497

Paired Samples Test


Pair 1 SCORE1 - SCORE2 1.271 20 .218

Pair 2 SCORE1 - SCORE3 3.283 20 .004

Pair 3 SCORE1 - SCORE4 -7.932 20 .000

Pair 4 SCORE2 - SCORE3 2.048 20 .054

Pair 5 SCORE2 - SCORE4 -6.745 20 .000

Pair 6 SCORE3 - SCORE4 -8.131 20 .000







Anti-Mia

T-Test



Std. Error

Mean

SCORE1 10.53 30 7.583 1.384 Pair 1

SCORE2 11.80 30 8.256 1.507

SCORE1 10.53 30 7.583 1.384 Pair 2

SCORE3 5.40 30 3.359 .613

SCORE1 10.53 30 7.583 1.384 Pair 3

SCORE4 9.20 30 7.708 1.407

SCORE2 11.80 30 8.256 1.507 Pair 4

SCORE3 5.40 30 3.359 .613

SCORE2 11.80 30 8.256 1.507 Pair 5

SCORE4 9.20 30 7.708 1.407

SCORE3 5.40 30 3.359 .613 Pair 6

SCORE4 9.20 30 7.708 1.407


N Correlation Sig.









Anti-Mia (Continued)

Paired Samples Test

Paired Differences

95% Confidence

Interval of the

Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 -1.267 3.723 .680 -2.657 .123

Pair 2 SCORE1 - SCORE3 5.133 6.329 1.155 2.770 7.496

Pair 3 SCORE1 - SCORE4 1.333 6.200 1.132 -.982 3.648

Pair 4 SCORE2 - SCORE3 6.400 6.425 1.173 4.001 8.799

Pair 5 SCORE2 - SCORE4 2.600 7.668 1.400 -.263 5.463

Pair 6 SCORE3 - SCORE4 -3.800 7.694 1.405 -6.673 -.927

Paired Samples Test


Pair 1 SCORE1 - SCORE2 -1.864 29 .073

Pair 2 SCORE1 - SCORE3 4.443 29 .000

Pair 3 SCORE1 - SCORE4 1.178 29 .248

Pair 4 SCORE2 - SCORE3 5.456 29 .000

Pair 5 SCORE2 - SCORE4 1.857 29 .073

Pair 6 SCORE3 - SCORE4 -2.705 29 .011







Anti-Jka

T-Test



Std. Error

Mean

SCORE1 19.20 10 10.185 3.221 Pair 1

SCORE2 17.80 10 8.548 2.703

SCORE1 19.20 10 10.185 3.221 Pair 2

SCORE3 11.50 10 8.606 2.721

SCORE1 19.20 10 10.185 3.221 Pair 3

SCORE4 24.30 10 8.564 2.708

SCORE2 17.80 10 8.548 2.703 Pair 4

SCORE3 11.50 10 8.606 2.721

SCORE2 17.80 10 8.548 2.703 Pair 5

SCORE4 24.30 10 8.564 2.708

SCORE3 11.50 10 8.606 2.721 Pair 6

SCORE4 24.30 10 8.564 2.708


N Correlation Sig.









Anti-Jka (Continued)

Paired Samples Test

Paired Differences

95% Confidence Interval

of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 1.400 3.204 1.013 -.892 3.692

Pair 2 SCORE1 - SCORE3 7.700 3.561 1.126 5.153 10.247

Pair 3 SCORE1 - SCORE4 -5.100 5.301 1.676 -8.892 -1.308

Pair 4 SCORE2 - SCORE3 6.300 3.466 1.096 3.821 8.779

Pair 5 SCORE2 - SCORE4 -6.500 3.866 1.222 -9.265 -3.735

Pair 6 SCORE3 - SCORE4 -12.800 4.662 1.474 -16.135 -9.465

Paired Samples Test


Pair 1 SCORE1 - SCORE2 1.382 9 .200


Pair 3 SCORE1 - SCORE4 -3.042 9 .014


Pair 5 SCORE2 - SCORE4 -5.317 9 .000

Pair 6 SCORE3 - SCORE4 -8.683 9 .000







Anti-Jkb

T-Test



Std. Error

Mean

SCORE1 20.10 10 9.803 3.100 Pair 1

SCORE2 18.40 10 8.409 2.659

SCORE1 20.10 10 9.803 3.100 Pair 2

SCORE3 8.90 10 6.190 1.958

SCORE1 20.10 10 9.803 3.100 Pair 3

SCORE4 23.80 10 11.144 3.524

SCORE2 18.40 10 8.409 2.659 Pair 4

SCORE3 8.90 10 6.190 1.958

SCORE2 18.40 10 8.409 2.659 Pair 5

SCORE4 23.80 10 11.144 3.524

SCORE3 8.90 10 6.190 1.958 Pair 6

SCORE4 23.80 10 11.144 3.524


N Correlation Sig.









Anti-Jkb (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 1.700 3.164 1.001 -.563 3.963

Pair 2 SCORE1 - SCORE3 11.200 6.339 2.004 6.666 15.734

Pair 3 SCORE1 - SCORE4 -3.700 4.498 1.422 -6.918 -.482

Pair 4 SCORE2 - SCORE3 9.500 4.927 1.558 5.975 13.025

Pair 5 SCORE2 - SCORE4 -5.400 5.168 1.634 -9.097 -1.703

Pair 6 SCORE3 - SCORE4 -14.900 8.762 2.771 -21.168 -8.632

Paired Samples Test




Pair 3 SCORE1 - SCORE4 -2.601 9 .029


Pair 5 SCORE2 - SCORE4 -3.304 9 .009

Pair 6 SCORE3 - SCORE4 -5.378 9 .000







Anti-Fya

T-Test



Std. Error

Mean

SCORE1 29.86 7 17.459 6.599 Pair 1

SCORE2 26.14 7 19.152 7.239

SCORE1 29.86 7 17.459 6.599 Pair 2

SCORE3 13.86 7 14.983 5.663

SCORE1 29.86 7 17.459 6.599 Pair 3

SCORE4 17.71 7 15.478 5.850

SCORE2 26.14 7 19.152 7.239 Pair 4

SCORE3 13.86 7 14.983 5.663

SCORE2 26.14 7 19.152 7.239 Pair 5

SCORE4 17.71 7 15.478 5.850

SCORE3 13.86 7 14.983 5.663 Pair 6

SCORE4 17.71 7 15.478 5.850


N Correlation Sig.









Anti-Fya (Continued)

Paired Samples Test

Paired Differences

95% Confidence

Interval of the

Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 3.714 3.251 1.229 .707 6.721

Pair 2 SCORE1 - SCORE3 16.000 5.416 2.047 10.991 21.009

Pair 3 SCORE1 - SCORE4 12.143 6.817 2.577 5.838 18.448

Pair 4 SCORE2 - SCORE3 12.286 8.056 3.045 4.835 19.737

Pair 5 SCORE2 - SCORE4 8.429 9.589 3.624 -.440 17.297

Pair 6 SCORE3 - SCORE4 -3.857 2.410 .911 -6.086 -1.628

Paired Samples Test







Pair 6 SCORE3 - SCORE4 -4.234 6 .005







Anti-Fyb

T-Test



Std. Error

Mean

SCORE1 23.00 5 15.116 6.760 Pair 1

SCORE2 22.00 5 13.638 6.099

SCORE1 23.00 5 15.116 6.760 Pair 2

SCORE3 12.20 5 8.786 3.929

SCORE1 23.00 5 15.116 6.760 Pair 3

SCORE4 19.80 5 8.955 4.005

SCORE2 22.00 5 13.638 6.099 Pair 4

SCORE3 12.20 5 8.786 3.929

SCORE2 22.00 5 13.638 6.099 Pair 5

SCORE4 19.80 5 8.955 4.005

SCORE3 12.20 5 8.786 3.929 Pair 6

SCORE4 19.80 5 8.955 4.005


N Correlation Sig.









Anti-Fyb (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 1.000 6.364 2.846 -6.902 8.902

Pair 2 SCORE1 - SCORE3 10.800 9.576 4.283 -1.090 22.690

Pair 3 SCORE1 - SCORE4 3.200 8.167 3.652 -6.941 13.341

Pair 4 SCORE2 - SCORE3 9.800 7.694 3.441 .246 19.354

Pair 5 SCORE2 - SCORE4 2.200 5.119 2.289 -4.156 8.556

Pair 6 SCORE3 - SCORE4 -7.600 3.647 1.631 -12.128 -3.072

Paired Samples Test


Pair 1 SCORE1 - SCORE2 .351 4 .743





Pair 6 SCORE3 - SCORE4 -4.660 4 .010







Anti-Dia

T-Test



Std. Error

Mean

SCORE1 16.80 5 14.755 6.598 Pair 1

SCORE2 19.20 5 15.849 7.088

SCORE1 16.80 5 14.755 6.598 Pair 2

SCORE3 10.20 5 9.445 4.224

SCORE1 16.80 5 14.755 6.598 Pair 3

SCORE4 23.00 5 16.340 7.308

SCORE2 19.20 5 15.849 7.088 Pair 4

SCORE3 10.20 5 9.445 4.224

SCORE2 19.20 5 15.849 7.088 Pair 5

SCORE4 23.00 5 16.340 7.308

SCORE3 10.20 5 9.445 4.224 Pair 6

SCORE4 23.00 5 16.340 7.308


N Correlation Sig.









Anti-Dia (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 -2.400 3.130 1.400 -6.287 1.487

Pair 2 SCORE1 - SCORE3 6.600 6.914 3.092 -1.985 15.185

Pair 3 SCORE1 - SCORE4 -6.200 5.167 2.311 -12.616 .216

Pair 4 SCORE2 - SCORE3 9.000 7.348 3.286 -.124 18.124

Pair 5 SCORE2 - SCORE4 -3.800 3.564 1.594 -8.225 .625

Pair 6 SCORE3 - SCORE4 -12.800 8.526 3.813 -23.387 -2.213

Paired Samples Test


Pair 1 SCORE1 - SCORE2 -1.714 4 .162


Pair 3 SCORE1 - SCORE4 -2.683 4 .055


Pair 5 SCORE2 - SCORE4 -2.384 4 .076

Pair 6 SCORE3 - SCORE4 -3.357 4 .028







Anti-E+c

T-Test�



Std. Error

Mean

SCORE1 20.10 10 20.642 6.528 Pair 1

SCORE2 18.80 10 19.612 6.202

SCORE1 20.10 10 20.642 6.528 Pair 2

SCORE3 10.70 10 14.697 4.648

SCORE1 20.10 10 20.642 6.528 Pair 3

SCORE4 26.80 10 24.068 7.611

SCORE2 18.80 10 19.612 6.202 Pair 4

SCORE3 10.70 10 14.697 4.648

SCORE2 18.80 10 19.612 6.202 Pair 5

SCORE4 26.80 10 24.068 7.611

SCORE3 10.70 10 14.697 4.648 Pair 6

SCORE4 26.80 10 24.068 7.611


N Correlation Sig.









Anti-E+c (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 1.300 3.743 1.184 -1.378 3.978

Pair 2 SCORE1 - SCORE3 9.400 11.157 3.528 1.418 17.382

Pair 3 SCORE1 - SCORE4 -6.700 4.572 1.446 -9.970 -3.430

Pair 4 SCORE2 - SCORE3 8.100 12.556 3.971 -.882 17.082

Pair 5 SCORE2 - SCORE4 -8.000 5.077 1.606 -11.632 -4.368

Pair 6 SCORE3 - SCORE4 -16.100 14.985 4.739 -26.819 -5.381

Paired Samples Test




Pair 3 SCORE1 - SCORE4 -4.634 9 .001


Pair 5 SCORE2 - SCORE4 -4.983 9 .001

Pair 6 SCORE3 - SCORE4 -3.398 9 .008







Anti-C+e

T-Test�



Std. Error

Mean

SCORE1 25.60 5 26.425 11.818 Pair 1

SCORE2 24.80 5 28.102 12.567

SCORE1 25.60 5 26.425 11.818 Pair 2

SCORE3 10.60 5 10.945 4.895

SCORE1 25.60 5 26.425 11.818 Pair 3

SCORE4 24.00 5 32.086 14.349

SCORE2 24.80 5 28.102 12.567 Pair 4

SCORE3 10.60 5 10.945 4.895

SCORE2 24.80 5 28.102 12.567 Pair 5

SCORE4 24.00 5 32.086 14.349

SCORE3 10.60 5 10.945 4.895 Pair 6

SCORE4 24.00 5 32.086 14.349


N Correlation Sig.









Anti-C+e (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 .800 5.215 2.332 -5.676 7.276

Pair 2 SCORE1 - SCORE3 15.000 17.219 7.701 -6.380 36.380

Pair 3 SCORE1 - SCORE4 1.600 11.437 5.115 -12.601 15.801

Pair 4 SCORE2 - SCORE3 14.200 19.422 8.686 -9.915 38.315

Pair 5 SCORE2 - SCORE4 .800 11.009 4.923 -12.870 14.470

Pair 6 SCORE3 - SCORE4 -13.400 21.582 9.652 -40.198 13.398

Paired Samples Test







Pair 6 SCORE3 - SCORE4 -1.388 4 .237







Anti-E+Mia

T-Test



Std. Error

Mean

SCORE1 16.63 8 19.078 6.745 Pair 1

SCORE2 19.75 8 20.817 7.360

SCORE1 16.63 8 19.078 6.745 Pair 2

SCORE3 9.13 8 8.676 3.067

SCORE1 16.63 8 19.078 6.745 Pair 3

SCORE4 15.25 8 7.440 2.631

SCORE2 19.75 8 20.817 7.360 Pair 4

SCORE3 9.13 8 8.676 3.067

SCORE2 19.75 8 20.817 7.360 Pair 5

SCORE4 15.25 8 7.440 2.631

SCORE3 9.13 8 8.676 3.067 Pair 6

SCORE4 15.25 8 7.440 2.631


N Correlation Sig.









Anti-E+Mia (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 -3.125 2.295 .811 -5.044 -1.206

Pair 2 SCORE1 - SCORE3 7.500 10.954 3.873 -1.658 16.658

Pair 3 SCORE1 - SCORE4 1.375 15.080 5.332 -11.232 13.982

Pair 4 SCORE2 - SCORE3 10.625 12.648 4.472 .051 21.199

Pair 5 SCORE2 - SCORE4 4.500 16.639 5.883 -9.411 18.411

Pair 6 SCORE3 - SCORE4 -6.125 6.105 2.158 -11.229 -1.021

Paired Samples Test


Pair 1 SCORE1 - SCORE2 -3.851 7 .006





Pair 6 SCORE3 - SCORE4 -2.838 7 .025







Anti-E+Jka

T-Test



Std. Error

Mean

SCORE1 23.67 9 13.314 4.438 Pair 1

SCORE2 23.56 9 15.043 5.014

SCORE1 23.67 9 13.314 4.438 Pair 2

SCORE3 10.11 9 6.716 2.239

SCORE1 23.67 9 13.314 4.438 Pair 3

SCORE4 30.89 9 18.489 6.163

SCORE2 23.56 9 15.043 5.014 Pair 4

SCORE3 10.11 9 6.716 2.239

SCORE2 23.56 9 15.043 5.014 Pair 5

SCORE4 30.89 9 18.489 6.163

SCORE3 10.11 9 6.716 2.239 Pair 6

SCORE4 30.89 9 18.489 6.163


N Correlation Sig.









Anti-E+Jka (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 .111 4.567 1.522 -3.400 3.622

Pair 2 SCORE1 - SCORE3 13.556 10.163 3.388 5.744 21.367

Pair 3 SCORE1 - SCORE4 -7.222 8.729 2.910 -13.932 -.513

Pair 4 SCORE2 - SCORE3 13.444 10.584 3.528 5.309 21.580

Pair 5 SCORE2 - SCORE4 -7.333 4.743 1.581 -10.979 -3.687

Pair 6 SCORE3 - SCORE4 -20.778 13.479 4.493 -31.139 -10.417

Paired Samples Test




Pair 3 SCORE1 - SCORE4 -2.482 8 .038


Pair 5 SCORE2 - SCORE4 -4.638 8 .002

Pair 6 SCORE3 - SCORE4 -4.624 8 .002







Anti-S+Mia

T-Test



Std. Error

Mean

SCORE1 10.60 5 3.435 1.536 Pair 1

SCORE2 13.20 5 6.686 2.990

SCORE1 10.60 5 3.435 1.536 Pair 2

SCORE3 9.40 5 4.159 1.860

SCORE1 10.60 5 3.435 1.536 Pair 3

SCORE4 14.60 5 5.273 2.358

SCORE2 13.20 5 6.686 2.990 Pair 4

SCORE3 9.40 5 4.159 1.860

SCORE2 13.20 5 6.686 2.990 Pair 5

SCORE4 14.60 5 5.273 2.358

SCORE3 9.40 5 4.159 1.860 Pair 6

SCORE4 14.60 5 5.273 2.358


N Correlation Sig.









Anti-S+Mia(Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 -2.600 3.912 1.749 -7.457 2.257

Pair 2 SCORE1 - SCORE3 1.200 .837 .374 .161 2.239

Pair 3 SCORE1 - SCORE4 -4.000 1.871 .837 -6.323 -1.677

Pair 4 SCORE2 - SCORE3 3.800 3.701 1.655 -.796 8.396

Pair 5 SCORE2 - SCORE4 -1.400 3.286 1.470 -5.481 2.681

Pair 6 SCORE3 - SCORE4 -5.200 1.304 .583 -6.819 -3.581

Paired Samples Test


Pair 1 SCORE1 - SCORE2 -1.486 4 .211


Pair 3 SCORE1 - SCORE4 -4.781 4 .009


Pair 5 SCORE2 - SCORE4 -.953 4 .395

Pair 6 SCORE3 - SCORE4 -8.918 4 .001







Anti-E+c+Mia

T-Test



Std. Error

Mean

SCORE1 23.36 11 10.433 3.146 Pair 1

SCORE2 22.55 11 11.579 3.491

SCORE1 23.36 11 10.433 3.146 Pair 2

SCORE3 10.55 11 4.865 1.467

SCORE1 23.36 11 10.433 3.146 Pair 3

SCORE4 23.55 11 10.501 3.166

SCORE2 22.55 11 11.579 3.491 Pair 4

SCORE3 10.55 11 4.865 1.467

SCORE2 22.55 11 11.579 3.491 Pair 5

SCORE4 23.55 11 10.501 3.166

SCORE3 10.55 11 4.865 1.467 Pair 6

SCORE4 23.55 11 10.501 3.166


N Correlation Sig.









Anti-E+c+Mia (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 .818 4.215 1.271 -2.013 3.650

Pair 2 SCORE1 - SCORE3 12.818 7.083 2.135 8.060 17.576

Pair 3 SCORE1 - SCORE4 -.182 10.255 3.092 -7.071 6.708

Pair 4 SCORE2 - SCORE3 12.000 8.355 2.519 6.387 17.613

Pair 5 SCORE2 - SCORE4 -1.000 12.562 3.788 -9.439 7.439

Pair 6 SCORE3 - SCORE4 -13.000 8.683 2.618 -18.834 -7.166

Paired Samples Test



Pair 2 SCORE1 - SCORE3 6.002 10 .000

Pair 3 SCORE1 - SCORE4 -.059 10 .954

Pair 4 SCORE2 - SCORE3 4.764 10 .001

Pair 5 SCORE2 - SCORE4 -.264 10 .797

Pair 6 SCORE3 - SCORE4 -4.965 10 .001







Anti-E+c+Jka

T-Test



Std. Error

Mean

SCORE1 23.50 10 14.175 4.483 Pair 1

SCORE2 21.40 10 12.085 3.822

SCORE1 23.50 10 14.175 4.483 Pair 2

SCORE3 9.70 10 7.675 2.427

SCORE1 23.50 10 14.175 4.483 Pair 3

SCORE4 28.50 10 17.703 5.598

SCORE2 21.40 10 12.085 3.822 Pair 4

SCORE3 9.70 10 7.675 2.427

SCORE2 21.40 10 12.085 3.822 Pair 5

SCORE4 28.50 10 17.703 5.598

SCORE3 9.70 10 7.675 2.427 Pair 6

SCORE4 28.50 10 17.703 5.598


N Correlation Sig.









Anti-E+c+Jka (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 2.100 4.701 1.487 -1.263 5.463

Pair 2 SCORE1 - SCORE3 13.800 8.011 2.533 8.069 19.531

Pair 3 SCORE1 - SCORE4 -5.000 9.381 2.966 -11.711 1.711

Pair 4 SCORE2 - SCORE3 11.700 7.009 2.216 6.686 16.714

Pair 5 SCORE2 - SCORE4 -7.100 9.433 2.983 -13.848 -.352

Pair 6 SCORE3 - SCORE4 -18.800 12.118 3.832 -27.469 -10.131

Paired Samples Test




Pair 3 SCORE1 - SCORE4 -1.685 9 .126


Pair 5 SCORE2 - SCORE4 -2.380 9 .041

Pair 6 SCORE3 - SCORE4 -4.906 9 .001







Combined two Ab

T-Test



Std. Error

Mean

SCORE1 19.18 39 17.650 2.826 Pair 1

SCORE2 19.67 39 18.025 2.886

SCORE1 19.18 39 17.650 2.826 Pair 2

SCORE3 9.95 39 9.464 1.516

SCORE1 19.18 39 17.650 2.826 Pair 3

SCORE4 22.62 39 19.485 3.120

SCORE2 19.67 39 18.025 2.886 Pair 4

SCORE3 9.95 39 9.464 1.516

SCORE2 19.67 39 18.025 2.886 Pair 5

SCORE4 22.62 39 19.485 3.120

SCORE3 9.95 39 9.464 1.516 Pair 6

SCORE4 22.62 39 19.485 3.120


N Correlation Sig.









Combined two Ab (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 -.487 4.192 .671 -1.846 .872

Pair 2 SCORE1 - SCORE3 9.231 11.254 1.802 5.583 12.879

Pair 3 SCORE1 - SCORE4 -3.436 9.555 1.530 -6.533 -.338

Pair 4 SCORE2 - SCORE3 9.718 12.124 1.941 5.788 13.648

Pair 5 SCORE2 - SCORE4 -2.949 10.052 1.610 -6.207 .310

Pair 6 SCORE3 - SCORE4 -12.667 13.735 2.199 -17.119 -8.214

Paired Samples Test


Pair 1 SCORE1 - SCORE2 -.726 38 .472

Pair 2 SCORE1 - SCORE3 5.122 38 .000

Pair 3 SCORE1 - SCORE4 -2.246 38 .031

Pair 4 SCORE2 - SCORE3 5.006 38 .000

Pair 5 SCORE2 - SCORE4 -1.832 38 .075

Pair 6 SCORE3 - SCORE4 -5.759 38 .000







Combined three Ab

T-Test



Std. Error

Mean

SCORE1 23.87 23 11.849 2.471 Pair 1

SCORE2 22.91 23 11.528 2.404

SCORE1 23.87 23 11.849 2.471 Pair 2

SCORE3 11.17 23 6.939 1.447

SCORE1 23.87 23 11.849 2.471 Pair 3

SCORE4 26.70 23 14.452 3.013

SCORE2 22.91 23 11.528 2.404 Pair 4

SCORE3 11.17 23 6.939 1.447

SCORE2 22.91 23 11.528 2.404 Pair 5

SCORE4 26.70 23 14.452 3.013

SCORE3 11.17 23 6.939 1.447 Pair 6

SCORE4 26.70 23 14.452 3.013


N Correlation Sig.









Combined three Ab (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 .957 4.557 .950 -1.014 2.927

Pair 2 SCORE1 - SCORE3 12.696 7.413 1.546 9.490 15.901

Pair 3 SCORE1 - SCORE4 -2.826 9.666 2.015 -7.006 1.354

Pair 4 SCORE2 - SCORE3 11.739 7.225 1.507 8.615 14.863

Pair 5 SCORE2 - SCORE4 -3.783 11.123 2.319 -8.593 1.027

Pair 6 SCORE3 - SCORE4 -15.522 10.587 2.207 -20.100 -10.944

Paired Samples Test


Pair 1 SCORE1 - SCORE2 1.007 22 .325

Pair 2 SCORE1 - SCORE3 8.214 22 .000

Pair 3 SCORE1 - SCORE4 -1.402 22 .175

Pair 4 SCORE2 - SCORE3 7.792 22 .000

Pair 5 SCORE2 - SCORE4 -1.631 22 .117

Pair 6 SCORE3 - SCORE4 -7.031 22 .000







Total Ab

T-Test



Std. Error

Mean

SCORE1 23.77 175 17.789 1.345 Pair 1

SCORE2 19.01 175 13.866 1.048

SCORE1 23.77 175 17.789 1.345 Pair 2

SCORE3 11.59 175 9.902 .749

SCORE1 23.77 175 17.789 1.345 Pair 3

SCORE4 18.84 175 15.447 1.168

SCORE2 19.01 175 13.866 1.048 Pair 4

SCORE3 11.59 175 9.902 .749

SCORE2 19.01 175 13.866 1.048 Pair 5

SCORE4 18.84 175 15.447 1.168

SCORE3 11.59 175 9.902 .749 Pair 6

SCORE4 18.84 175 15.447 1.168


N Correlation Sig.

Pair 1 SCORE1 & SCORE2 175 .769 .000

Pair 2 SCORE1 & SCORE3 175 .833 .000

Pair 3 SCORE1 & SCORE4 175 .470 .000

Pair 4 SCORE2 & SCORE3 175 .776 .000

Pair 5 SCORE2 & SCORE4 175 .757 .000

Pair 6 SCORE3 & SCORE4 175 .518 .000



Total Ab (Continued)

Paired Samples Test

Paired Differences


of the Difference

Mean

Std. Deviation

Std. Error

Mean

Lower Upper

Pair 1 SCORE1 - SCORE2 4.760 11.366 .859 3.064 6.456

Pair 2 SCORE1 - SCORE3 12.171 10.997 .831 10.531 13.812

Pair 3 SCORE1 - SCORE4 4.926 17.223 1.302 2.356 7.495

Pair 4 SCORE2 - SCORE3 7.411 8.786 .664 6.101 8.722

Pair 5 SCORE2 - SCORE4 .166 10.319 .780 -1.374 1.705

Pair 6 SCORE3 - SCORE4 -7.246 13.349 1.009 -9.237 -5.254

Paired Samples Test


Pair 1 SCORE1 - SCORE2 5.540 174 .000

Pair 2 SCORE1 - SCORE3 14.642 174 .000

Pair 3 SCORE1 - SCORE4 3.783 174 .000

Pair 4 SCORE2 - SCORE3 11.159 174 .000

Pair 5 SCORE2 - SCORE4 .212 174 .832

Pair 6 SCORE3 - SCORE4 -7.180 174 .000







BIOGRAPHY

NAME Miss Kallaya Kirdkoungam

DATE OF BRITH 25 November 1971

PLACE OF BRITH Samutprakran, Thailand

INSTITUTIONS ATTENDED Chulalongkron University, 1991-1994:

Bachelor of Science

(Genetics)

Mahidol University, 2005-2007:

Master of Science

(Transfusion Science)

RESEARCH GRANT Siriraj Grant for Research Development

Faculty of Medicine, Siriraj Hospital

Mahdol University

POSITION & OFFICE 1997-Present, Antiserum and Standard Cells

Preparation Section. National Blood Centre,

Thai Red Cross Society, Henri Dunant Road.

Pathumwan, Bankok, Thailand.

Tel (02)2524106-9 Ext.1706

E-mail: [email protected]
