Monitoring Alloantibodies in Sickle Cell Disease Patients

This study focuses on monitoring the frequency and effects of alloantibodies in sickle cell disease patients after multiple blood transfusions. It aims to analyze the prevalence of alloantibodies and provide knowledge for the medical industry to deal with the issue. The study will use a retrospective cohort study and collect data from regional medical centers.

Title: Monitoring alloantibodies for the patients of
sickle cell disease after the multiple blood transfusion
Name:
Student’s id:
Date:

1. Project title
Monitoring alloantibodies for the patients of sickle cell disease after the multiple
blood transfusion
2. Abstract
Introduction The frequency of alloiimmunization to the red blood cells of the sickle
cell anaemia patients is increasing at an alarming rate. Multiple antibodies among the
patients’ body is the source of multiple health complications. Blood transfusion is a
common therapeutic tool used in treating the patients of sickle cell disease. Due to
frequent transfusions, various complications among the patients can be found. Among
the complications, alloimmunization is a life threatening issue which is occurred due
to irregular RBC antibodies. It is associated with various other risks of human body
that can lead to the death of the patient (Makarovska-Bojadzieva, Velkova and
Blagoevska2017). However, the risk of alloimmunization can be decreased by
following preventive measures.
Aim The study is focused to analyze the facts regarding blood transfusion and
alloimmunization.
Objectives Primary objective will be to study the low frequency antibodies and its
effects on the health of the human beings. It will also find the process of dealing with
the issue. It will also determine the prevalence of alloantibodies.
Significance of the study It will provide wider information and knowledge so that
medical industry can have ideas to deal with the issue.
Ethics Written consent from the institutional review board will be obtained to avoid
ethical issues as confidential record of the patients will be used in the study.
Materials and Methods A retrospective cohort study will be used to collect and
analyze data in this regard. Sample will be obtained from a particular regional medical

centres and non parametric statistical analysis method will be used to analyse them.
However, there will be various limitations of the study, but proper measures will be
taken in order to minimize those limitations. The budget as well as the timing of the
whole study will also be taken into account while preparing the research proposal.
3. Introduction
3.1 General introduction
Sickle cell anaemia is a kind of disorder related to red blood cells. It means, if
someone is dealing issues regarding the haemoglobin and its functionality then there
is a chance that he/she is suffering from sickle cell anaemia. Haemoglobin is basically
a kind of protein that is present in the red blood cells of the human body and oxygen
throughout the body (Piel, Steinberg and Rees 2017). In sickle cell disorder, the
haemoglobins are formed into stiff rods in the red blood cells which in turn change the
actual shape of the red blood cells and from the shape of a disc it is changed into a
sickle or crescent shape (Ware et al. 2017). Transfusion of the red blood cells is a very
common and effective intervention in treating and preventing complications
associated with sickle cell disease. It has been found in a report that the incidence of
RBC alloimmunization among the sickle cell anaemia patients range between 8% to
50% and the average range is 20% to 30%. Chronic transfusion therapy is being used
among the patients of sickle cell anaemia and the frequency has also been increased a
lot. In order to prevent morbidity and mortality, the demand for the blood transfusion
has been increased. Several studies clearly indicated the importance of providing
antigen-matched blood for the patients of chronic blood transfusion such as sickle cell
anaemia. It helps in reducing the frequency of alloimmunization and the multiple
complications related to it. Many patients found to be developed alloantibodies which
are responsible for various health complications. Aiming to increase the tissue

delivery of oxygen, blood transfusion is preferred by the physicians. In some cases,
patients do not require much blood transfusion but there are many cases, where a
frequent blood transfusion is much needed. This multiple blood transfusion
contributes to the multiple types of health complications that include
alloimmunization (Zalpuri et al. 2017). With the advancement of technology and
increased rate of life expectancy across the world, the number of complex surgeries
and chronic degenerative diseases has been increased and it requires a very high
number of blood transfusions as the treatment, and thus, the frequency of
alloantibody, not belonging to the ABO system are increasing (Estcourt et l. 2017). As
a consequence, it promotes the higher risk of delayed haemolytic transfusion reactions
(Stotesbury et al. 2018). Additionally, the investigations regarding RBC
alloimmunizations are generally performed after the transfusion events. Over the last
few years, it has been found in various researches that irregular RBC antibodies are
associated with the majority of fatal haemolytic transfusion reaction which is one of
the leading causes of the deaths due to transfusions (Bhuva and Vachhani 2017).
3.2 Aim/Statement of the problem
It has been clearly analyzed by the researchers that alloantibody can be the reason of
delayed haemolytic transfusion reactions and among the sickle cell disease patients it
can trigger hyperhemolysis in which patients’ blood cells are destroyed and takes the
patient one step ahead to the death. It is also one of the biggest reasons behind the
disappearance of positive antibodies screening. All these facts provided a direct
motivation towards the study and hence, the aim of the study is to have a detailed
analysis on the facts regarding this issue found among the patients from all over the
world.

3.3 Purpose/Objectives
Considering the statement of the problem, the main and primary objective of this
paper is to study the low frequency antibodies present in the sickle cell anaemia
which can be easily detected through antibody screening test. Along with this, the
study is also highly focused to identify the specific antibodies and its effects on
the human body. At the same time it will also analyze how to deal with such
complications to live a better life. Another prime objective of this study is to
determine the prevalence of red blood cells alloantibodies among the patients with
blood transfusion. The study will be organized among the patients of Saudi Arabia
and in this regard, hospitals and healthcare organizations of Saudi Arabia will be
considered.
3.4 Significance of the study
The study that will be performed is quite significant for the benefit of the patients
as well as the medical industry. With the increased life expectancy and number of
major operations, blood transfusion has become one of the most common parts in
the treatment procedure but it is a leading cause of complications like
alloimmunization. This study will analyse the antibodies among the patients of
sickle cell anaemia as in this disease multiple blood transfusion is required as the
procedure of treatment. This paper will also help people to understand the process
of dealing with alloantibodies by determining the prevalence of alloantibodies in
the red blood cells after the multiple blood transfusions. This study will open an
eye of knowledge about the issue and it will also help medical industry to set
proper or any alternative treatment to avoid issues in this matter.
3.5 Literature review

Alloimmunization to the RBC antigen is mainly one of the major complications of
chronic blood transfusion among the sickle cell anaemia patients. It may require
some immunosuppressive drug, any alternative method and others. But,
preventing such complication is most necessary than curing the disease. Blood
transfusion is a source of multiple health complications which may be life
threatening and in this regard researchers conducted multiple surveys and studies.
Various studies and researches have been conducted by the researchers regarding
the topic of blood transfusion and its association with the alloimmunization.
Transfusion of the red blood cells is considered as one of the essential therapeutic
tools in treating the patients of sickle cell disease. But a repeated blood transfusion
is a leading cause of alloimmunization that causes difficulty in cross-matching. A
study was conducted among the Palestinians to investigate the frequency of RBC
alloimmunization among the patients of sickle cell disease who undergo multiple
blood transfusions. A multicenter cross-sectional study was conducted in this
regard among 116 transfused patients of SCD and it was found that the frequency
of RBC among the alloimmunization patients was comparatively low. However,
the rate among the Palestinian SCD patients is comparatively low than any other
countries but still it requires attention (Samarah et al. 2018). Again, another study
was conducted among Omani patients with sickle cell disease to evaluate the red
blood cell alloimmunization. 262 patients participated in the study and it was
found in the result that the rate of alloimmunization among the SCD patients was
31.6%. The study concluded that the red blood cells transfusions is directly
associated with the alloimmunization among the patients. Also, the higher
incidence of the alloimmunization is among the patients of SCD. The study also
produced the result that the rate of the alloimmunization found among the Omani

patients was almost similar to the patients of other countries (Alkindi et al. 2017).
According to various literatures and studies, red blood cell transfusions are mainly
the cornerstone in treating and preventing sickle cell disease. This is generally
applied either as an episodic treatment or as a chronic transfusion scheme among
the patients as per their requirements. RBC alloimmunization is a common as well
as serious complication in the transfusion therapy. A study was organized in this
regard aiming to assess the risk of first alloimmunization from the from the first
lifetime transfusion and that study was also focused to find the individual risks
associated with the alloimmunization with varying gender, haemoglobin genotype,
age and other factors. A retrospective, multicentre, new user cohort study was
organized by the researchers among the patients of various care centres in
Netherlands. After collecting data the researchers statistically analysed them and
in the result it was found that, 1012 SCD patients were having abnormalities in
their haemoglobin. Comparing the results with the other general population it was
clearly found that patients with sickle cell disease who undergo frequent blood
transfusion are at high risk of alloimmuniztaion than that of the generally
transfused patients. The study also demonstrated that the risk of alloimmunization
also increases if the number of blood transfusion is increased among the sickle cell
disease patients. The study also concluded that the first transfusion after the age of
5 is strongly associated with the risks of alloimmunization. Again, exposure to the
episodic transfusion also takes one step closer to the risk of such complications
due to multiple blood transfusion process among the sickle cell disease patients.
The authors in the literature finally put an end to their report by stating that only
an early identification of the patients with alloimmunization will be extremely
helpful in enabling a cost-effective approach to prevent and cure alloimmunization

among the patients. Literatures stated that the pathogen of alloimmunization is not
really well-understood. As a result of RBC transfusion the risk of
alloimmunization is quite high among the patients of sickle cell disease who need
repeated blood transfusions as their therapeutic tool. This alloimmunization can be
result in various health complications that may lead to life-threatening episodes of
haemolysis as well haemolytic diseases of the fetus and new born. The authors in
their study analyzed the incidence as well as the complications of
alloimmunization among the SCD patients. The approaches to mitigate the
complications were also reviewed in their analysis. The authors reviewed various
literatures from EMBASE and MEDLINE and other databases that were written
from 2006 to 2016 as their research method. The result was yielded from 168
resources and it was found in the result that the rate of alloimmunization among
the SCD patient is 30%. However, the study also suggested that various biological
factors impact the risk of alloimmunization. Patients with SCD can develop
hyperhemolysis which is a critical condition that can lead to the destruction of the
RBCs of both recipients and the donors. The authors mentioned in their paper that
in order to prevent alloimmunization, serologic matching program is considered as
a successful method compared to others because in this program antisera to
phenotype blood donor is used. However, in a recently published economic
analysis, it has also been found by the researchers that the cost of providing
serologically similar RBCs to patients is quite high among the SCD patients. A
true quantity of cost is hard to define, and thus the authors of the literature
provided an estimation that a preventing a single alloimmunization will cost
$369,482 over 10 years (Gehrie et al. 2017). Other authors from various literatures
also exposed the fact that multiple blood transfusions are the leading causes of

various health complications that are life-threatening and it includes delayed
haemolytic transfusion reactions (DHTR) and it may occur within 24 hours up to
21 days after the blood transfusion. The mismatch of RBC antigen is one of the
major reasons behind the occurrence of alloimmunization among the sickle cell
disease patients. Again, children have been found to have lower alloimmunization
when compared to older adults who undergo multiple blood transfusions. The
alloantibodies which is produced by the recipient is the key cause of DHTR and
these alloantibodies were undetectable prior to the transfusion. The
alloimmunization promotes alloantibody formation which is comparatively higher
among the sickle cell disease patients. However, the authors suggested that a good
documentation of antibody history can be helpful enough as a preventive strategy
of alloimmunization after the blood transfusion among the patients of sickle cell
anaemia. Transfusion from ethnically matched donor is required in this regard as
suggested by the authors. However, almost all the authors suggested further
research in order to obtain a more appropriate as well as an accurate result.
3.6 Hypothesis/ Research question
After having a thorough research on the background of the issues as well the main
problem statement of this paper, the research question has been set. This study
will directly focus to find the answer of the main research question which in turn
will fulfil all the requirements to achieve the main objective of the study. The
research question will emphasize the findings from the literatures and gaps or the
problems identified in the literature review. Specific alloantibodies can create life
threatening issues and thus the main research question is,
A. How low frequency antibodies affect the health of patients with multiple
blood transfusions?

B. How to deal with it?
4. Materials and methods
Materials and methods is the most essential part of any research. This section of the
paper will provide all the technical details of the proposed experiments and hence, the
design, setting, sample, procedure and ethical board approval will be discussed in
detail.
4.1 Design
Various types of methods and designs are there to complete any kind of study or
research. In retrospective cohort studies, people have already developed the
outcome of interest and the investigators jump back to the past to identify the
cohort of individuals even when the outcome of interest was not found. This kind
of study is very much efficient to study the rare as well as unusual exposures.
Therefore, to find the answer of the research questions for this study, a
retrospective multicentre cohort study method will be designed. On the basis of
the report, all the exposed and unexposed record of the patients will be found in
this study. The population of the study will be from a particular region, Riyadh
and the sample size will be 100. It will be a lengthy process and hence, it will take
around one year to be completed.
4.2 Setting
Most of the sample in this research will be collected from the King Saud medical
City and the Blood Bank Central at the regional laboratory of Riyadh. If a patient
will have positive antibody screening test or any positive previous history on
sickle cell disease will be considered as a part of the study. The study is
retrospective cohort and thus the sample size will not be broad enough. From
those locations, 100 samples will be collected who matched the selection criteria

and they will be chosen as the participants of the study regarding
alloimmunization and blood transfusion.
4.3 Sample
The study population has been chosen from the same region and different centres
will be selected in this regard to find the sample size required for this study. The
patients, whose background or history on the blood transfusion will not be found,
will be excluded from the study. Also, the patients who carried antibodies before
blood transfusion will be excluded from the study. The patients, who matched the
selection criteria, will only be chosen as a part of the proposed study and also, the
group of participants will be irrespective of their biological factors such a
genotype, age and any others. A random sampling method will be followed.
Sample size is calculated using power analysis method, after analyzing the
hypothesis, effect size and variability. There will be 5 different steps in this
regard. At first, the main hypothesis will be chosen and explicit in terms of null.
Then a significance level of the test will be specified. The smallest effect size will
also be specified. The value of the other parameters will be estimated. Finally, the
intended power of the test will be specified.
4.4 Procedure
Data consisting information about the health of the patients will be collected from
the laboratories of the hospital and the medical centre chosen. All documented
RBC units and full information along with the background history of the patients
will be collected. Hospital authority will be contacted if any further information is
required in this regard. For various kinds of experiments we will use IH1000 and
ABO and Ortho-Clinical Diagnostics machine. Database from the previous
publications will also be used to compare the result of the cohort study. It is

assumed that the procedure that will be followed in the study will be successful
enough in answering the hypothesis as well as the research questions. A set of
questionnaires will be asked to the patients to get direct review from them and
their responses will be recorded in computers.
4.5 Institutional Review Board (IRB)/Ethical Board Approval
In order to avoid any kind of ethical and legal risk and to gain benefit of the study,
it is important to protect the confidentiality of the participants as their personal
data will be collected. Due to this, it is important to have their permission and so,
a written consent will be obtained from the Institutional Review for the approval
of the study. Initially application document package will be assembled and signed
off by the members doing the research. Then it will be forwarded to the Ethical
Board for approval. Reviewing the application notification of the approval will be
sent. The main benefit is, in this way, all kind of ethical issues regarding the
confidentiality of the patients can be avoided. There will be no opportunity of any
third party misuse for the confidential data of the patients which is a major risk.
5. Statistical Analyses
Non-parametric tests are more robust than the parametric tests and here, the sample size is
also small and hence, a non-parametric statistical analysis method will be chosen to analyse
the data collected. Again, the result found in the nonparametric statistical analysis method
will be valid in a broader range of situations. Here, we will use IH1000 and ABO and
Ortho-Clinical Diagnostics machine to analyse the data. The SPSS software
contains variety of tools to analyse data and thus that will be used in this
study.
6. Limitations of the study

The issue regarding the alloimmunization and the complications related to it is important in
clinical management. It is also highly necessary for the laboratory practice among the sickle
cell anaemia patients. This study will be focussed onto this issue and patients of Saudi Arabia
will be considered as the participants in this regard. There can be some limitations of this
study, as will collect data from a particular region only. Also, as the study will be
retrospective in nature, the population will consist of patients who are exposed to various
transfusion protocols over time. Another limitation of the study is the long time interval
between the blood transfusion and the screening of the antibodies responsible for
alloimmunization. Therefore, it is recommended that a 7-days post transfusion screening
method can be used to minimize such limitations in this study.

7. References
Alkindi, S., AlMahrooqi, S., AlHinai, S., AlMarhoobi, A., Al-Hosni, S., Daar, S.,
Fawaz, N. and Pathare, A., 2017. Alloimmunization in patients with sickle cell
disease and thalassemia: Experience of a single centre in Oman. Mediterranean
journal of hematology and infectious diseases, 9(1).
Estcourt, L.J., Fortin, P.M., Hopewell, S., Trivella, M. and Wang, W.C., 2017. Blood
transfusion for preventing primary and secondary stroke in people with sickle cell
disease. Cochrane Database of Systematic Reviews, (1).
Bhuva, D.K. and Vachhani, J.H., 2017. Red cell alloimmunization in repeatedly
transfused patients. Asian journal of transfusion science, 11(2), p.115.
Gehrie, E.A., Ness, P.M., Bloch, E.M., Kacker, S. and Tobian, A.A., 2017. Medical
and economic implications of strategies to prevent alloimmunization in sickle cell
disease. Transfusion, 57(9), pp.2267-2276.
Makarovska-Bojadzieva, T., Velkova, E. and Blagoevska, M., 2017. The impact of
extended typing on red blood cell alloimmunization in transfused patients. Open
access Macedonian journal of medical sciences, 5(2), p.107.
Piel, F.B., Steinberg, M.H. and Rees, D.C., 2017. Sickle cell disease. New England
Journal of Medicine, 376(16), pp.1561-1573.
Samarah, F., Srour, M.A., Yaseen, D. and Dumaidi, K., 2018. Frequency of Red
Blood Cell Alloimmunization in Patients with Sickle Cell Disease in
Palestine. Advances in hematology, 2018.

Stotesbury, H., Kirkham, F.J., Kölbel, M., Balfour, P., Clayden, J.D., Sahota, S.,
Sakaria, S., Saunders, D.E., Howard, J., Kesse-Adu, R. and Inusa, B., 2018. White
matter integrity and processing speed in sickle cell anemia. Neurology, 90(23),
pp.e2042-e2050.
Ware, R.E., de Montalembert, M., Tshilolo, L. and Abboud, M.R., 2017. Sickle cell
disease. The Lancet, 390(10091), pp.311-323.
Zalpuri, S., Evers, D., Zwaginga, J.J., Schonewille, H., de Vooght, K.M., le Cessie, S.
and van der Bom, J.G., 2017. and alloimmunization against red blood cell
transfusions. clinical determinants of red cell alloimmunization, 54(8), p.97.

8. Budget
There will be government funding for this research project and no
other private institution will be associated with this project for any
financial purpose.
9. Time Table
Data will be collected after the blood transfusion and occurrence of
alloimmunization, after that the data will be analysed and due to
this purpose the whole project will be time consuming and it will
take near about a year to get completed.

Appendices
Questionnaires
1. How many times you undergo blood transfusion?
2. When the alloimmunization was first detected?
3. What were the symptoms?
4. Did you undergo blood transfusion for any other reason than
sickle cell disease?