Diet and ABO Antibody Titer 1

CHAPTER I
INTRODUCTION

BACKGROUND OF THE STUDY

An antibody titer refers to the strength of an antibody, measured by determining the
greatest dilution of antibody-containing serum that will produce a detectable reaction with a
standard volume of red blood cells possessing the corresponding antigen (Lynch and Raphael,
1983).
Antibodies serve as body’s defense system that fights sickness and infection wherever
they are needed. Increased levels of antibodies can help a person feel better and get sick less
often while low levels of antibodies usually indicate a medical problem such as infection or a
recent treatment(Davis, 2011). There are many factors that affect the antibody production such as
medications or eating the right foods. This study focuses on the effect of dietary intake of food to
the level of antibody titer.
As Harmening (2012) describes, ABO antibodies are naturally-occurring because they
are produced without any exposure to RBC antigens. These are predominantly IgM, there may be
small quantities of IgG present, and reacts at room temperature or colder.ABO antibodies in the
serum are formed naturally, in response to ABO blood group antigens that are not found on the
individual’s RBCs. (Dean, 2005). The production of ABO antibodies are initiated at birth but are
too low for detection until the individual is 3 to 6 months of age. ABO antibody production
peaks when an individual has reached the age between 5 and 10 years and declines later, but
slowly, in life. Elderly people usually have lower anti-A and anti-B antibodies that are
undetectable in the reverse grouping. In cases where blood transfusion is needed, ABO
antibodies can cause intravascular hemolysis when the wrong blood unit is transfused, leading to
the death of the patient (Harmening, 2012). The ABO system contains four major ABO
phenotypes: A, B, O & AB. These antibody phenotypes produce strong direct agglutination
reactions during ABO testing procedures such as ABO blood typing. Serum from group O
individuals contains not only anti-A and anti-B but also anti-A,B, which reacts with A and B
cells. Anti-A,B antibody activity, originally thought to be just a mixture of anti-A and anti-B,
cannot be separated into a pure specificity when adsorbed with either A or B cells. For example,
if group O serum is adsorbed with A or B cells, the antibody eluted will react with both A and B
cells. Anti-A, B therefore possesses serologic activity not found in mixtures of anti-A plus anti-
B(Harmening, 2012).
Individuals produce anti-A and anti-B of the IgM class. Some, particularly those who are
group O, also produce IgG antibodies. A-like and B-like antigens found in different substances
such as food, in the environment or in other living organisms, such as microbes, seem to
stimulate the production of these antibodies. On the other hand, when someone has IgG anti-A,B,
the amount of IgM anti-A,B is usually high and the term high-titer anti-AB or high-titer O is
used. The serum of these individuals will often lyse A and/or B cells in the reverse grouping.
These high-titre antibodies are important in two situations (High Titre Anti A/B Testing of
Donors within the National Blood Services (NBS), 2002).
Furthermore high-titer ABO antibodies can be observed in group O multiparous women
and in patients taking certain bacteria-based nutritional supplements. Although older reports
indicated a fall in isoagglutinin titers in the elderly, more recent studies have disputed those
findings. In industrialized countries, isoagglutinin titers have decreased with increasing
consumption of processed foods (Cooling, 2011).
Diet and ABO Antibody Titer 2

Antibody titration is a determination of the concentration of a specific antibody in the

patient's serum or to determine the strength of antigen expression on different red cell samples. 
If the concentration of the specific antibody is being determined, the cells must contain the
known antigen and the procedure should be performed under the optimal conditions for that
antibody (AABB Technical Manual, 14th Ed, 2003).The usual applications of titration studies are
estimating the antibody activity of an alloimmunized pregnant female, attempting to determine if
there is any specificity to an autoantibody and characterizing antibodies that may be high titer,
low avidity antibodies. (AABB Technical Manual, 14th Ed., 2003).
For a diet to accomplish its function, it has to contain the essential macro- and
micronutrients, especially those that mediate immune function. It is basic knowledge that the
food groups already contain these important elements of nutrition. In a micronutrient research
conducted in a school in the United States, the inadequate intake of these nutrients can definitely
lead to immune deficiency then, ultimately, susceptibility to infection and disease. Protein-
Calorie malnutrition is the number one most common nutritional problem in the world today.
The study further states that antibody affinity and response to pathogens is decreased in this type
of malnutrition (Drake, 2010).
The diet that we take also does not only encompass the things we eat but also the way we
take in our food. A study was made by Johnson, Kudsk, Fukatsu, Renegar and Zarzaur (2003) to
assess if the lack of enteral feeding significantly impairs the generation of immune responses to
an acute viral infection and this has shown that antibody-forming cells have significantly
decreased in the 13-day period allotted for the experiment. The study was concluded that there
was a significant decrease of mucosal IgA due to lack of enteric stimulation. In relation to the
study at hand, maximal antibody production was achieved as the diet was not parenterally
introduced but enterically stimulated, which has led to the values of the antibody titers seen in
the tables above.
Blood and blood products are used for a number of purposes, but the three main reasons
for blood transfusion are to correct anemia, replace blood loss by bleeeding, either during
surgery or accident, and to replace other constituents of blood, such as coagulation factors.
(Blood Group Serology, 2009) It is essential that before blood be transfused, the donor blood
must be compatible with the blood of the recipient, that ABO compatibility must be achieved.
Considerations such that AB individuals are universal recipients and O individuals as universal
donors must be put into practice, to avoid transfusion reactions. For pregnant women, it must be
noted if maternal serum lyses A and/or B cells as IgG anti-A,B could have crossed the placenta
and destroyed the fetal red cells. The baby, upon birth may suffer from the effects of anemia and
jaundice as a result of red cell destruction, though ABO Hemolytic Disease of the Fetus and
Newborn (HDFN) is less severe, but more common than Rh HDFN. Quantifying the level of
antibodies at this point, therefore, does not have any value in performing any further tests during
pregnancy.
Probiotics modulate your immune system, an effect that has an impact not only on cancer
but also on your overall health status. Probiotics can restore and rebalance the gut microbiome,
strengthening its ability to interact with your immune system in many ways. These friendly
bacteria stimulate healthy immune surveillance, boosting populations of cells that seek out and
destroy infecting organisms and cancers. They inflammatory cytokines are upregulated during
the acute stage of an infection, cancer, or other threat to your body’s integrity, but they also
contribute to suppression of the inflammatory response as the infection dissipates (Killian, 2012).
Diet and ABO Antibody Titer 3

Vaccination initiates exposure to controlled amounts of antigens that elicit an immune

response, whether in responsive or non-responsive populations. Probiotic vaccination studies
have shown enhanced antibody titers, lower percentages of non-seroconverters and greater
percentages reaching minimum cut-off titer values in healthy adults, elderly and children. These
results indicate that probiotics are a good candidate to stimulate responses to vaccines and thus,
according to EFSA, enhance the function of the immune system related to defense against
infection. (West, P. N and Cripps, A. W. 2013)
Raw and processed food antibodies against their antigens were detected in a recent study
made by Vojdani (2009). Compared to raw food antigens, IgE antibodies showed a significant
increase against processed food antigens in a vast majority of the patients. Similarly, IgG, IgA
and IgM antibodies against modified food antigens overall were found at much higher levels than
antibody reactions against raw food antigens. Almost every tested serum with high levels of
antibodies against modified food antigens showed very high levels of antibodies against myelin
basic protein, oxidized low density lipoprotein, AGE-human serum albumin and AGE-
hemoglobin.
Generally, when a person eats the right foods, it can increase the level of its antibodies
and has the same effect as medications only without the side effects (Davis, 2011). Processed
foods is defined as any food other than a raw agricultural commodity, including any raw
agricultural commodity that has been subject to washing, cleaning, milling, cutting, chopping,
heating, pasteurization, blanching, cooking, canning, freezing, drying, dehydrating, mixing,
packaging, or other procedures that alter the food from its natural side. Processing also may
include the addition of other ingredients to the food, such as preservatives, flavors, nutrients, and
other food additives or substances approved for use in food products, such as salt, sugars, and
fats. Processing foods, including the addition of ingredients, may reduce, increase, or leave
unaffected the nutritional characteristics of raw agricultural commodities. [(US Department of
Health and Human Services, Dietary Guidelines Advisory Committee 2010)]

Objectives of the Study

The study aims to determine the effect of diet on the ABO antibody titer of the
respondents. Specifically, it seeks to:
1. Which among the respondents prefer the following aside from their usual diet:
a. Bacterial-based products (Probiotic)
b. Multi-vitamins
c. Processed food products
d. Raw foods
e. All kinds of food mentioned.

2. Which among the group has the highest ABO antibody titer?

3. Is there significant difference in the ABO antibody titer of the respondents when
grouped as to their diet?

4. Is there a significant difference in the ABO antibody titer of the respondents between
their diet?
Diet and ABO Antibody Titer 4

Theoretical Framework
According to the 15th edition of the AABB Technical Manual (2005), individuals that do
not have the A and/or B antigens will produce an antibody against the absent antigen. This
indicates a relationship that permits the predictability of the ABO testing of sera and of red cells.
The development of antibodies is based on the fact that the configurations that confer A and B
antigenic determinants also exist in other biological entities such as bacteria cell walls. Their
presence in environment, intestinal flora, dust, food and other widely distributed agents, ensures
a constant exposure of all persons to A-like or B-like antigens.
In addition, in the report of Schley and Field (2002) the gastrointestinal tract is subjected
to enormous and continual foreign antigenic stimuli from food and microbes. This organ must
integrate complex interactions among diet, external pathogens, and local immunological and
non-immunological processes. It is critical that protective immune responses are made to
potential pathogens, while hypersensitivity reactions to dietary antigens are minimized. There is
increasing evidence that fermentable dietary fibers and the newly described probiotics can
modulate various properties of the immune system, including those of the gut-associated
lymphoid tissues (GALT). Changes in the intestinal microflora that occur with the consumption
of probiotic fibers may potentially mediate immune changes via the direct contact of lactic acid
bacteria or bacterial products--such as the cell wall or cytoplasmic components--with immune
cells in the intestine, the production of short-chain fatty acids from fiber fermentation, or by
changes in mucin production.
Goljan (2013) mentions that these blood group antibodies are made in Peyer’s patches,
specifically located in the GIT. Located there are specialized epithelial cells called M (microfold)
cells that overlie these Peyer’s patches and trap the incoming A and or B antigens. Once trapped,
the M cells transport these antigens to lymphocytes in the Peyer’s Patches, resulting in the
development of natural antibodies against the antigens from the food. He also adds that natural
antibodies develop against antigens that are not present on the RBC, which explains why the
blood group O individuals have both anti-A and anti-B.
According to the study by Thomsen and Kettel that dates back to 1929, as cited by Auf
Der Maur, Hodel, Nydegger, and Reiben (1993) on the reexamination of the ABO histo-blood
group antibodies, ABO antibodies in the decrease in agglutination titers with the increasing age
of the individuals was far less pronounced than previously described. According to American
Association of Blood Banks Technical Report (2011), isoagglutinin titers in industrialized
countries have generally decreased with increasing consumption of processed foods.
Moreover, animal studies have shown that animal kept in a sterile room from birth do not
produce antibodies. This is an indication that antibody production results from substances, which
abound in nature (Erhabor and Adias, 2013).

Significance of the Study

The major aim of the study is to determine the effect of diet on ABO antibody titers,
especially on the application of blood banking and transfusion practices. There is much
speculation regarding the consumption of food and its relationship with the increase and decrease
of ABO antibodies, especially their existing isoagglutinin titers. Since processed foods are
considered to be one of the major consumption of people today it is also important to consider
some important factors like food allergy, intolerance and sensitivity.
Diet and ABO Antibody Titer 5

It was concluded in different researches that the determination of food allergy,

intolerance and sensitivity would be improved by testing IgE, IgG, IgA and IgM antibodies
against both raw and processed food antigens. Antibodies against modified food antigens, by
reacting with AGEs and tissue proteins, may cause perturbation in degenerative and autoimmune
diseases such as diabetes, atherosclerosis, inflammation, autoimmunity, neurodegeneration and
neuro-autoimmunity.
If such claims are proven, ABO antibodies in relation to transfusion practices could be
vital in a way that screening methods could be refined even more to the point that diet of such
person is considered as one of the major factors for blood donation. Also, such quantitative
measurement could also be a determinant of one's nutritional health aside from other tests that
could be done in the laboratory.
Fellow medical technologists, nutritionists, doctors and food and drug scientists could be
feasible targets for this research as it will aid them in their further research on the abilities of the
immune system to react and produce antibodies in response to diet-induced antigenic reactions.
Nutritionists could also come up with ways on formulating feasible food additives that
eliminates the risk of acquiring allergic reactions. Also, food processing could be redefined in a
way that it could give healthier and health-oriented without losing the essence of the food. If this
research were to be utilized, it would supply them with essential research material that would aid
them in their search for solutions to food allergy reactions in the body. The antibodies produced
against antigens acquired from raw and/or processed food could serve as markers for diet-related
illnesses and become part of serologically routine tests in the future.
Diet and ABO Antibody Titer 6

CHAPTER II

METHODOLOGY

Research Design

This study utilized the cross-sectional approach of research. In a cross-sectional study, all
the measurements are made at about the same time, with no follow-up period (Hulley, 2007).
The effect of diet in the ABO antibody titer was determined involving participants within the age
range of 16-19 with a diet that suited to the criteria provided by the researchers. The researchers
also provided food products that augmented the diet needed in order to obtain consistency.
The specific research problems were answered by the data gathered through a structured
survey questionnaires assess and group the respondents as to their preferred food group. After
gathering the results of the survey and grouping the respondents, the researchers determined the
ABO antibody titer of the respondents, and statistical analysis was performed.

Population and Sampling Plan

The respondents of the study were 51 tertiary students of private higher education
institution in Baguio City for the Second Semester, S.Y. 2013 2014. To determine the
respondents of the study, convenient sampling was applied taking ten (10) respondents per
dietary preference group. Grouping of the respondent was done by taking the most preferred diet
of the respondent. An Informed Consent Form (Appendix B) was attached in order to fulfil the
legal and ethical requirements for research studies at the Saint Louis University, Baguio City. It
is used to inform about the purpose of the study and for the patient to comprehend the rationale
of blood extraction and its possible adverse reaction to the respondents and its corresponding
action and management. After signing the informed consent and agreeing to participate in the
study, 5ml of venous blood was extracted from the respondents and determination of ABO
antibody titer was followed.

Methods

Data Gathering Tools

Survey Questionnaire. Survey questionnaire was used to determine the frequency of the
consumption of foods of the respondents aside from their usual diet. It is in the form of
checklists and rating scales that contain probable answers to questions for the respondents to
select (See Appendix B). It consists of items that include the different types of food such as
bacterial based products (probiotics), multivitamins, processed foods, raw foods and the
combination of all the foods mentioned. For each group of diet, a brief definition and
examples of food was provided. In order to measure the frequency of consumption, a 5-point
scale was used:
5- Always (Daily)
4- Often (5-6 times a week)
3- Sometimes (3-4 times a week)
2- Seldom (1-2 times a week)
1- Never (0- no consumption)
Diet and ABO Antibody Titer 7

Grouping of the respondents was done by taking the most preferred diet group of
the respondent, wherein the group of diet with the highest frequency such as “5- Always”
and “4- Often” was considered as a basis in grouping the respondent.

ABO Antibody Titration

Accordingly, ABO antibody titration was performed. Prior to titration procedure,
the ABO blood type of the participant was determined by means of Reverse Typing or
Serum Typing which determine the type of antibody present in the participant’s serum.
The procedure for ABO antibody titration was adopted from the American
Association of Blood Banks Technical Manual 15th Edition (2005) (See Appendix D)
with modification. The test was performed in the saline phase only, thus detecting only
naturally occurring antibodies which are IgM in nature.

Data Analysis

The data that was gathered was tabulated and computed using the Statistical
Package for Social Sciences (SPSS) Software.
Frequency count and percentage was used to determine which among the
provided food groups is preferred by the respondents aside from their usual diet.
Kruskal-Wallis Test was employed to determine the significant differences on the
ABO antibody titer of the respondents when grouped as to their preferred food groups
aside from their usual diet. This non parametric test compared the ABO antibody titer of
the respondents among the five (5) group of diet. A P-value of less than 0.01 was
considered statistically significant.
To determine the significant difference lies, post hoc Mann-Whitney U test was
employed to analyze the significant differences in the ABO antibody titer of the
respondents between the groups of diet. A P-value of less than 0.01 was considered
statistically significant.
Diet and ABO Antibody Titer 8

Chapter III
Results and Discussion

This chapter deals with the analysis and discussion of the data gathered on the effect of
diet in the ABO antibody titer of the respondents as well as the significant differences on the
ABO antibody titer between and among the different groups of diet.

Distribution of the respondents as to their preferred diet.

Figure 1. Distribution of the respondents as to their preferred diet.

Combination of all four

20%
Probiotics
20%

Raw Food Multivitamins

20% 20%

Processed
22%

ABO antibody titer of the respondents as to their diet

Table 1. Mean Rank of ABO Antibody titers

Diet of the Respondents
Probiotics Processed Multi- Raw Mixed
vitamins
Mean Rank 51.57 21.13 29.68 41.67 38.70

Table 1 presents the mean rank of the respondents’ antibody titer using the Kruskal-
Wallis test. This means that ABO antibody titer of the respondents under the probiotic group has
the highest ABO antibody titer among the five groups of diet. On the other hand, the ABO
antibody titer of the respondents under the processed food group has the least ABO antibody titer
among the five groups of diet.
Diet and ABO Antibody Titer 9

Significant difference in the ABO antibody titer of the respondents among their diet

Table 2. Summary table of Kruskal Wallis Analysis of the ABO antibody titer

Summary Table
Kruskal-Wallis H value 19.421
Df 4
Asymp. Sig. .001
α=0.01

Kruskal-Wallis test was conducted to evaluate the significant difference in the ABO
antibody titer of the respondents then grouped as to their diet. As presented on table 2, the test
revealed significant difference in the ABO antibody titer of the respondents on to their diet
(χ2=19.421, p=0.01); the mean ranks of the different groups of diet are significantly different
among them. Because the overall test is significant, pairwise comparisons among the five groups
of diet was conducted.
Significant difference in the ABO antibody titer of the respondents between their diet

Table 4. Pairwise comparison between the groups of diet using Mann-Whitney U Test.

Diet Asymp. Sig. Interpretation

(2-tailed)
Probiotics vs. Multivitamins .002 Significant
Probiotics vs. Raw .142 Not Significant
Probiotics vs. Processed .000 Significant
Probiotics vs. Mix .095 Not Significant
Multivitamins vs. Raw .088 Not Significant
Multivitamins vs. Processed .111 Not Significant
Multivitamins vs. Mix .222 Not Significant
Raw vs. Processed .007 Significant
Raw vs. Mix .764 Not Significant
Processed vs. Mix .023 Not Significant
(α=0.01 )

Mann-Whitney U test was used to determine the significant difference between the
groups of diet. Pairwise comparisons that exhibit significant difference mean that when two
groups are being compared, one group exhibits a higher antibody titer than the other group.
While, comparisons that exhibit no significant difference means that the ABO antibody titer of
the groups compared have nearly the same level of antibody titer.
It was found out that the individuals that consumed bacterial-based products had the
highest level of antibody titer among the different groups of diet. The antibody titer levels in this
study depended on the type of diet the individuals consumed. This confirms the concept provided
by AABB that biological entities, such as bacteria, contain the configuration that make up their
A-like and/or B-like antigenic determinants, causing a raise in antibody titers.
Diet and ABO Antibody Titer 10

Consumption of bacterial-based products confer the greatest effect when it comes to

immune system reinforcement. Probiotics are known to be immune modulators that constantly
stimulate the immune system to “be on guard” against potentially harmful pathogens (Cross,
2006). These friendly bacteria stimulate healthy immune surveillance, boosting populations of
cells that seek out and destroy infecting organisms and cancers. Their inflammatory cytokines
are upregulated during the acute stage of an infection, cancer, or other threat to your body’s
integrity, but they also contribute to suppression of the inflammatory response as the infection
dissipates (Killian, 2012).
The other diets used in the study, such as processed food, raw food, mixed food diet and
multivitamins, presented the values that succeeded the probiotic diet. Raw and processed food
antibodies against their antigens were detected in a recent study made by Vojdani (2009).
According to his study, processed food antigens significantly increased the value of
immunoglobulins in the body, as compared to raw food antigens. This study made by Vojdani
contradicts to the findings of the study conducted by the researchers, as revealed by the results of
the study stating that antibody titers of the individuals under the raw food group has a higher
level of antibody titer versus the antibody titer of the respondents who consumed processed food
products.
Finally, this study determined the effect of diet in the antibody titer of the respondents
and it was found out that probiotics has the greatest immune-stimulating potential as evidenced
by the high titer level of the probiotic food group.

REFERENCES
Diet and ABO Antibody Titer 11

Auf der Maur, C., Hodel, M., Nydegger, U.E., Rieben, R. (2007). Age dependency of
ABO histo-blood group antibodies: reexamination of an old dogma. Transfusion
(impact factor: 3.22). 33(11):915-8.

Blood Transfusion Safety (2006).Retrieved from

http://www.who.int/bloodsafety/en/Blood_Transfusion_Safety.pdf on 20 August
2013.

Blood Transfusion. (2008). Retrieved from http://www.transfusion.com.au., on 21 August

2013.

Cooling, L. (2011) ABO, H, and Lewis Blood Groups and Structurally Related

Antigens.American Association of Blood Banks Technical Manual 17th edition.

Daniels, G. (1995). Human blood groups. Oxford: Blackwell Science.

Davis, S. (2011). Foods That Increase Antibodies.

de França ND, Poli MC, Ramos PG, Borsoi CS, Colella R (2011). Titers of ABO
antibodies in group O blood donors.Rev Bras HematolHemoter. 2011; 33(4): 259–
262.

Dean, L. (2005). Blood groups and red cell antigens. Dean, Md.: NCBI.
epiCentral: Michigan Center for Public Health Preparedness. (n.d.). UM SPH - Office of
Public Health Practice. Retrieved October 14, 2013, from
http://practice.sph.umich.edu/micphp/epice

Erhabor and Adias (2013).Essentials of blood transfusion science. Central Milton

Keynes: Authorhouse.

Foods that increase antibodies.(2011). Retrieved from

http://www.livestrong.com/article/108418-foods-increse-antibpdis/ on 5
September 2013.

Guyton, A. C., & Hall, J. E. (2000). Textbook of medical physiology. Philadelphia:

Saunders.

Harmening, D. (2005). Modern blood banking and transfusion practices (5th ed.).

Philadelphia: F.A. Davis.

High Titre Anti-A/B Testing of Donors within the National Blood Services (NBS). (2002).
Retrieved from

Hulley, S. B. (2007). Designing clinical research (3rd ed.). Philadelphia, PA: Lippincott

Williams & Wilkins.ch. 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins,
2007.
Diet and ABO Antibody Titer 12

Lynch, M. J., & Raphael, S. S. (1983).Lynch's Medical laboratory technology(4th ed.).

Philadelphia: W.B. Saunders.

Mazda, T., Yabe, R., NaThalang, O., Thammayong, T., Tadokoro, K. (2007). Differences
in ABO antibody levels among blood donors: a comparison between past and
present Japanese, Laotian, and Thai populations. Japanese Red Cross Institute,
Tatsumi 2-1-67, Koto-ku, Tokyo 135-8521, Japan.

Protein and antibody production (1946). Nutrition Reviews, 4: 279–282.

doi: 10.1111/j.1753-4887.1946.tb08891.x

Schley, P.D. and Field, C.J. (2002).The immune-enhancing effects of dietary fibres and
prebiotics. British Journal of Nutrition . 87, Suppl. 2, S221–S230

Appendix A. LETTER TO THE DEAN

SAINT LOUIS UNIVERSITY

Diet and ABO Antibody Titer 13

A. BONIFACIO STREET, 2600 BAGUIO CITY, PHILIPPINES

S C H O O L O F N A TU R A L S C I EN C E S
Department of Medical Laboratory Science

15 October 2013

DR. GAUDELIA A. REYES

Dean, School of Natural Sciences
Saint Louis University
Baguio City

MADAM:

We, the undersigned third year Bachelor in Medical Laboratory Science students are
presently conducting our research study entitled: Diet and ABO Antibody Titer, in
partial fulfilment to the requirements in Medical Technology Research.

In view thereof, we would like to request your good office to allow us to conduct our
research which involves the third year BMLS Students for the second semester, SY 2013-
2014.

Your favorable action and approval to the request is crucial in maintaining the highest
scholarly standards and excellence of the University in terms ofexcellent missionary and
transformativeeducation along with building research culture among us.

Thank you and more power.

Very respectfully yours,

Valdez, Dennis Bryan A. Bangay, Mark Joseph Repoyo, Berlanne A.

Paltep , Rashell Anne C. Mendoza, Alliana Jean D. Pasion,Jared Kenan

S.

Noted:
Ms. Ann P. Opiña, RMT, MMSHM
Faculty Research Promoter

Recommending Approval:
Mrs. Jannette D. Awisan, RMT, MSMT
Department Head, Department of Medical Laboratory Science

Appendix B.INFORMED CONSENT

Diet and ABO Antibody Titer 14

SAINT LOUIS UNIVERSITY

A. BONIFACIO STREET, 2600 BAGUIO CITY, PHILIPPINES

S C H O O L O F N A TU R A L S C I EN C E S
Department of Medical Laboratory Science

INFORMED CONSENT

Research Title: Diet and ABO Antibody Titer

Researchers: Valdez, Dennis Bryan A.
Bangay, Marc Joseph R.
Pasion, Jared Kenan S.
Repoyo, Berlanne A.
Mendoza, Alliana Jean D.
Paltep, Rashell Anne C.
Opiña, Ann P.

Greetings!

You are being invited to participate in the study entitled “Diet and ABO Titer” under the
supervision of Ann P. Opiña,

The information provided on this form and the accompanying cover letter is presented to you in
order to fulfill the legal and ethical requirements for research studies at the Saint Louis
University, Baguio City.

Note that the following must have been explained well to you and you fully understand them
before you sign this consent form.

1. The purpose of this study is to assess the effect of diet on the ABO antibody titer of the
respondents. The knowledge gained from this study will guide the patients and also other
healthcare professionals about the effect of diet on the ABO antibody titer.

2. Participation is voluntary. Refusal to participate or withdrawal from the study will

present no penalty to participant.

3. The number of study participants is all BMLS third year students in Saint Louis
University.

4. The duration of the study will be two semester ( June 2013- March 2014).
Diet and ABO Antibody Titer 15

5. According to the researchers, reactions prior to blood extraction may include fear and
uncontrolled behavior; during the collection, pain and bleeding may occur. If it happens,
the researchers will not proceed immediately with the extraction or may stop the
extraction process and give further instructions for you to follow.

6. In case of adverse reactions, there will be immediate treatment/hospitalization which will

be available free charge. The researchers will provide reimbursement or payment for the
treatment and other expenses incurred because of the adverse event.

7. All your records or information will be kept strictly confidential.

8. You will be given information or results of the results of the tests that may be relevant to
your continued participation. You will also be informed of any new information which
may be relevant to your continued participation.

9. You can call or ask questions anytime regarding this study. The contact person for further
information or for consultation on diverse events is attached herewith.

10. Your signature on this consent form shows that you have been informed about the
conditions and safeguards of this project.

I have read the information provided and I agree to participate in this study

_________________________________
Signature of Participant, Date
Diet and ABO Antibody Titer 16

Appendix C.

SURVEY ON DIET PREFERENCE OF THE RESPONDENTS

Control No. _________

Please check the box that corresponds to the frequency of your consumption based on this 5-
point scale.

5 - Always (Daily)
4 - Often (5-6 times a week)
3 - Sometimes (3-4 times a week)
2 - Seldom (1-2 times a week)
FOOD TYPE 5
1 - Never (04 -no consumption)
3 2 1

Probiotic drinks

Multivitamins

Processed foods

Raw foods

Combination of all foods

mentioned
Diet and ABO Antibody Titer 17

Appendix C.

ABO ANTIBODY TITRATION

Principle
Titration is a semiquantitative method used to determine the concentration of antibody in
a serum sample or to compare the strength of antigen expression on different red cell samples.
The usual applications of titration studies are: 1) estimating antibody activity in alloimmunized
pregnant women to determine whether and when to perform more complex invasive
investigation of the fetal condition 2) elucidating autoantibody specificity 3) characterizing
antibodies as high-titer, low-avidity, traits common in antibodies to antigens of the Knops and
Chido/ Rodgers systems, Csa, and JMH and 4) observing the effect of sulfhydryl reagents on
antibody behavior, to determine immunoglobulin class (IgG or IgM).

Specimen
Serum or plasma antibody to be titrated.

Reagents
1. Red cells that express the antigen(s) corresponding to the antibody specificity (ies), in
a 2% to 5% saline suspension. Uniformity of cell suspensions is very important to ensure
comparability of results.
2. Saline. (Note: Dilutionsmay be made with albumin if desired.)

Procedure
The master dilution technique for titration studies is as follows:
1. Label 10 test tubes according to the serum dilution (eg, 1 in 1, 1 in 2, etc). A 1 in 1
dilution means one volume of serum undiluted; a 1 in 2 dilution means one volume of
serum in a final volume of two, or a 50% solution of serum in the diluent.
2. Deliver one volume of saline to all test tubes except the first (undiluted 1 in 1) tube.
3. Add an equal volume of serum to each of the first two tubes (undiluted and 1 in 2).
4. Using a clean pipette, mix the contents of the 1 in 2 dilution several times and transfer
one volume into the next tube (the 1 in 4 dilution).
5. Continue the same process for all dilutions, using a clean pipette to mix and transfer each
dilution. Remove one volume of diluted serum from the final tube and save it for use if
further dilutions are required.
6. Label 10 tubes for the appropriate dilutions.
7. Using separate pipettes for each dilution, transfer 2 drops of each diluted serum into the
appropriately labeled tubes and add 2 drops of a 2% red cell suspension. Alternatively,
for convenience, add 1 drop of a 3%-4% suspension of red cells as supplied by the
reagent manufacturer, although this method is less precise.
8. Mix well and test by a serologic technique appropriate to the antibody
9. Examine test results macroscopically; grade and record the reactions. The prozone
phenomenon may cause reactions to be weaker in the more concentrated serum
preparations than in higher dilutions. To avoid misinterpretation of results, it may be
preferable to examine first the tube containing the most dilute serum and proceed through
the more concentrated samples to the undiluted specimen.
Diet and ABO Antibody Titer 18

Interpretation
1. Observe the highest dilution that produces 1+ macroscopic agglutination. The titer is
reported as the reciprocal of the dilution level, eg, 32—not 1 in 32 or 1:32. If there is
agglutination in the tube containing the most dilute serum, the endpoint has not been
reached, and additional dilutions should be prepared and tested.
2. In comparative studies, a significant difference in titer is three or more dilutions.
Variations in technique and inherent biologic variability can cause duplicate tests to give
results that differ by one dilution in either direction. Serum containing antibody at a true
titer of 32 may show, on replicate tests, the endpoint in the 1:32 tube, the 1:64 tube, or the
1:16 tube.
3. Titer values alone can be misleading without also evaluating the strength of
agglutination. The observed strength of agglutination can be assigned a number and the
sum of these numbers for all tubes in a titration study represents the score, another
semiquantitative measurement of antibody reactivity. The arbitrarily assigned threshold
for significance in comparing scores is a difference of 10 or more between different test
samples.
4. Antibodies with high-titer, low-avidity characteristics generally have a titer greater than
64, with most tubes showing consistently weak reactivity.

Titration is a semiquantitative technique. Technical variables greatly affect the results and
care should be taken to achieve the most uniformpossible practices.

1. Careful pipetting is essential. Pipettes with disposable tips that can be changed after each
dilution are recommended.
2. Optimal time and temperature of incubation and time and force of centrifugation must be
used consistently.
3. The age, phenotype, and concentration of the test red cells will influence the results.
When the titers of several antibody-containing sera are to be compared, all of them
should be tested against red cells (preferably freshly collected) from the same donor. If
this is not possible, the tests should use a pool of reagent red cells from donors of the
same phenotype. When a single serum is to be tested against different red cell samples,
all samples should be collected and preserved in the same manner and diluted
to the same concentration before use.
4. Completely reproducible results are virtually impossible to achieve. Comparisons are
valid only when specimens are tested concurrently. In tests with a single serum against
different red cell samples, material from the master dilution must be used for all the tests.
5. Measurements are more accurate with large volumes than with small volumes; a master
dilution technique gives more reliable results than individual dilutions for a single set of
tests. The volume needed for all planned tests should be calculated and an adequate
quantity of each dilution prepared.
6. When performing a titration for anti-D for HDFN.

Reference:

Roback, J. D. (2005). Technical manual(15th ed.). Bethesda, Md.: AABB.

Diet and ABO Antibody Titer 19

Appendix D. TIMETABLE OF ACTIVITIES

ACTIVITY TIME FRAME REMARKS

Research Topic June 2013 Accomplished on June 26, 2013

Formulation
Research Proposal June-July 2013 Accomplished on July 17, 2013
Draft Making
Writing of July 2013 Submitted on August 14, 2013
Introduction
Revision of August 2013 Accomplished on September 18,
Introduction 2013
Writing of September 2013 Accomplished on October 9, 2013
Methodology and
Revisions
Proposal of October 2013 Submitted on October 18, 2013
Research/Submission
of Chapter 1 & 2
(as required for 1st
Semester)
Experimentation November –
December 2013
Data Gathering December 2013 To be done on Second Semester
Data Treatment December 2013 (SY 2013-2014)
Writing of Chapter 3 January 2013
Writing of Chapter 4 February 2013
Revisions and February 2013
Finalization of
research manuscript
Defense March 2013
Diet and ABO Antibody Titer 20

Appendix E. LINE-ITEM BUDGET

QUANTITY BUDGET ITEMS BUDGETARY

REQUIREMENT
1 pack (100pcs) Serum top (5ml) PHP 550.00
30 pcs Test Tubes PHP 350.00
8 pcs Tourniquet PHP 200.00
5 pcs Needle Adapter PHP 125.00
1 pack (1000 pcs) Pipette (Yellow) Tips PHP 350.00
50 pcs 2-way needle PHP 400.00
1 pack Absorbent cotton PHP 50.00
1 pc Micropore PHP 75.00
1 pc Labeling tape PHP 25.00
1 ream Bond paper (Letter) PHP 120.00
1 set Ink catriage (Black and Color) PHP 100.00
TOTAL PHP 2,345.00
Diet and ABO Antibody Titer 21