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BCR-ABL1 POSITIVE ACUTE LYMPHOBLASTIC LEUKAEMIA IN GHANAIAN 
PATIENTS 
 
 BY VICTOR OBENG OFORI  
STUDENT ID:  10550491 
 
THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN 
PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL 
HAEMATOLOGY DEGREE 
 
DEPARTMENT OF HAEMATOLOGY 
SCHOOL OF BIOMEDICAL AND ALLIED HEALTH SCIENCES 
COLLEGE OF HEALTH SCIENCES  
 
 
JULY 2018 
 
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DECLARATION 
I, Victor Obeng Ofori, a student of the Haematology Department, hereby make a declaration that 
this work is original and was carried out by me under the supervision by supervisors whose 
signatures are below.  
 Student:   Signature:    _____                            Date: 
 VICTOR OBENG OFORI 
 
Supervisors: 
Signature: _______________________________                         Date: 
AMMA ANIMA BENNEH-AKWASI KUMA – MBChB, FGCPS, FWACP                                                                           
Department of Haematology, University of Ghana                                                                                                                
 
 
Signature: ________________________________                        Date: 
EDEGHONGHON OLAYEMI – MSc., MBBS, FWACP                                                                 
Department of Haematology, University of Ghana                                                                                                                                         
 
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DEDICATION 
I dedicate this project to Almighty God for being my source of strength and wisdom and also to 
my dear mother, Vida Obeng Addai for her inspiration and support. I also dedicate this work to 
my dear wife, Rebecca Abora and my lovely daughter, Vida Obeng Addai Ofori. 
 
 
 
 
 
 
 
 
 
 
 
 
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ACKNOWLEDGEMENT 
I would like to express my profound gratitude to Professor Susan Crocker, Department of 
Pathology and Molecular Medicine at Queens University, Canada. Professor Crocker made this 
work possible by arranging for me to visit her laboratory at Queens to carry out the benchwork 
and reviewed this work. I would also like to thank Brooke Ring-Snetsinger of Queens 
Laboratory for Molecular Pathology (QLMP) for her patience and the time she devoted to train 
me on the fluorescent in-situ hybridisation technique.  I am also grateful to Shakeel Virk, Andy 
Zhang and all the other staff and students of QLMP for their assistance. 
I wish to give special thanks to my supervisors Dr. Amma Anima Benneh and Dr. Edeghonghon 
Olayemi of Haematology Department (University of Ghana) for their supervision and counseling 
which has enabled me complete this work. I would also like to thank the other lecturers in the 
Department (Prof. J. K. Acquaye and Dr. Yvonne Dei-Adomako) for their advice and 
encouragement.  To Francisco Torto and all the administrative and technical staff in the 
Haematology department who assisted me in various ways, I thank you. Many thanks to the staff 
of the Central laboratory (Haematology Special) and Haematology Day care unit of the Korle Bu 
Teaching Hospital. 
 I express my appreciation to Dr. Tom Ndanu (School of Medicine and Dentistry, University of 
Ghana) for his assistance in the statistical aspect of the work. Francis Krampa, Alice Charwudzi 
and all those who assisted me in diverse ways, I duly acknowledge your efforts. 
Finally, many thanks to Brosaman Company Ltd and Achimota Mile 7 Church of Christ who 
supported me with funds to be able to carry out this work.  
 
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TABLE OF CONTENTS 
 
DECLARATION ............................................................................................................................................... ii 
DEDICATION ................................................................................................................................................. iii 
ACKNOWLEDGEMENT .................................................................................................................................. iv 
TABLE OF CONTENTS ..................................................................................................................................... v 
LIST OF TABLES ............................................................................................................................................. ix 
LIST OF FIGURES ............................................................................................................................................ x 
ABBREVIATIONS ....................................................................................................................................... xi 
ABSTRACT ................................................................................................................................................xiii 
CHAPTER ONE ................................................................................................................................................ i 
INTRODUCTION .......................................................................................................................................... i 
1.1 Background ...................................................................................................................................... i 
1.2 Problem statement ......................................................................................................................... 3 
1.3 Justification ..................................................................................................................................... 4 
1.4 Aim .................................................................................................................................................. 5 
1.5 Specific Objectives .......................................................................................................................... 5 
CHAPTER TWO .............................................................................................................................................. 6 
LITERATURE REVIEW ................................................................................................................................. 6 
2.1 Definitions ....................................................................................................................................... 6 
2.2 Classification ................................................................................................................................... 7 
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2.3 Epidemiology ................................................................................................................................... 8 
2.4 Aetiology ......................................................................................................................................... 9 
2.5 Cytogenetics and Molecular genetics ........................................................................................... 10 
2.6 Risk Factors ................................................................................................................................... 11 
2.7 Pathogenesis ................................................................................................................................. 13 
2.8 Clinical features and laboratory findings ...................................................................................... 14 
2.9 Laboratory Diagnosis..................................................................................................................... 15 
2.10 Treatment ................................................................................................................................... 16 
2.11 Prognosis ..................................................................................................................................... 17 
2.12 Monitoring .................................................................................................................................. 18 
CHAPTER 3 .................................................................................................................................................. 19 
METHODOLOGY ...................................................................................................................................... 19 
3.1 Study design .................................................................................................................................. 19 
3.2 Study site ....................................................................................................................................... 19 
3.3 Study Population ........................................................................................................................... 20 
3.4 Inclusion criteria ............................................................................................................................ 20 
3.5 Exclusion criteria ........................................................................................................................... 20 
3.6 Sample size determination ........................................................................................................... 20 
3.7 Selection of samples and data collection ...................................................................................... 21 
3.8 Materials and Methods ................................................................................................................. 22 
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3.9 Procedure ...................................................................................................................................... 23 
3.10 Data Handling .............................................................................................................................. 27 
3.11 Statistical analysis ....................................................................................................................... 27 
CHAPTER 4 .................................................................................................................................................. 28 
RESULTS .................................................................................................................................................. 28 
4.1 Fluorescence In Situ Hybridization results and frequency of the BCR-ABL1 gene in samples. ..... 30 
4.2 Selected FISH Images .................................................................................................................... 31 
4.3 Demographics ............................................................................................................................... 36 
4.5 Descriptive and Inferential Statistics of Clinical features and BCR-ABL1 gene ............................. 38 
4.6 Descriptive and Inferential Statistics of Haematological parameters .......................................... 40 
4.7 Descriptive and Inferential Statistics of BCR-ABL1 gene and Clinical Outcome ........................... 41 
4.8 Features of the BCR-ABL1 Positive Cases ...................................................................................... 42 
CHAPTER 5 .................................................................................................................................................. 43 
DISCUSSION AND CONCLUSION .............................................................................................................. 43 
5.1 Frequency ...................................................................................................................................... 43 
5.2 Age and Gender ............................................................................................................................ 44 
5.3 Clinical Features ............................................................................................................................ 45 
5.4 Haematological Parameters .......................................................................................................... 45 
5.5 Clinical Outcome ........................................................................................................................... 47 
5.6 Limitations of the Study ................................................................................................................ 47 
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5.7 Conclusion ..................................................................................................................................... 47 
5.8 Recommendation .......................................................................................................................... 48 
REFERENCES ............................................................................................................................................ 49 
APPENDIX A ................................................................................................................................................. 61 
APPENDIX B ............................................................................................................................................. 64 
APPENDIX C ................................................................................................................................................. 68 
APPENDIX D ................................................................................................................................................. 69 
 
 
 
 
  
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LIST OF TABLES  
Table 1: IMMUNOPHENOTYPIC CLASSIFICATION OF ACUTE LYMPHOBLASTIC LEUKAEMIA
 ...................................................................................................................................................................... 7 
Table 2: DRUGS USED IN THE TREATMENT OF ACUTE LYMPHOBLASTIC LEUKAEMIA ....... 16 
Table 3: FISH RESULTS FOR BCR-ABL1 FUSION GENE ................................................................... 30 
Table 4: BCR-ABL1 FUSION GENE AND SEX ...................................................................................... 36 
Table 5: BCR-ABL1 FUSION GENE AND AGE ..................................................................................... 37 
Table 6: BCR-ABL1 FUSION GENE AND CLINICAL FEATURES ...................................................... 39 
Table 7: BCR-ABL1 FUSION GENE AND HAEMATOLOGICAL PARAMETERS ............................. 40 
Table 8: BCR-ABL1 FUSION GENE AND CLINICAL OUTCOME ...................................................... 41 
Table 9:  FEATURES OF BCR-ABL1 POSITIVE CASES ....................................................................... 42 
 
 
 
  
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LIST OF FIGURES 
Figure 1: FLOW DIAGRAM FOR THE SELECTION OF BONE MARROW ASPIRATE SLIDES FOR 
THE STUDY .............................................................................................................................................. 29 
Figure 2: Negative Control ......................................................................................................................... 31 
Figure 3: BCR-ABL1 fusion Negative Case ............................................................................................... 32 
Figure 4: Positive Control ........................................................................................................................... 33 
Figure 5:BCR-ABL1 fusion Positive Case ................................................................................................. 34 
Figure 6: BCR-ABL1 fusion case showing a single fusion signal .............................................................. 35 
 
 
 
 
 
 
 
 
 
 
 
 
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ABBREVIATIONS 
 
ABL1 –          Abelson murine leukaemia viral oncogene homolog 1 
AKT –           Ak strain transforming 
ALL -            Acute lymphoblastic leukaemia  
ATCC ® -        American Type Culture Collection 
BCR -             Breakpoint cluster region 
BCR-ABL1 -   Breakpoint cluster region - Abelson murine leukaemia viral oncogene homolog 1 
CDKN2A –    Cyclin-dependent kinase inhibitor 2A gene 
DAPI        -    4,6 – Diamidino v- 2- Phenylindole, Dihydrochloride) 
CDKN2B -     Cyclin-dependent kinase inhibitor 2B gene 
DNA –            Deoxyribonucleic acid  
E2A –              Transcription factor E2-alpha gene 
EBF1 -             Early B-cell factor 1 gene 
FISH –             Fluorescence in situ hybridisation 
GST T1-            Glutathione S transferase theta 1 gene 
ETV6-RUNX1 -ETS-variant 6 -runt-related transcription factor 1 gene 
Hb -                   Haemoglobin  
IL-7R –             Interleukin-7 receptor 
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IKZF1 -              Ikaros family zinc finger 1 gene 
JAK –                 Janus kinase  
KBTH -               Korle Bu Teaching Hospital 
MAPK  -            Mitogen-activated protein kinase  
MD -                   Monroe Dunaway 
mTor –                Mammalian target of rapamycin 
MTR – 5 –           methyl tetrahydrofolate-homocysteine methyltransferase gene  
MW -                  Mann-Whitney 
MLL-AF4 -          Mixed-lineage leukaemia -ALL-1 fused gene on chromosome 4 
PAX5 -                 Paired box 5 gene 
PBS –                 Phosphate buffered saline 
PI3 –                   Phosphatidylinositol -4,5- bisphosphate 3 - kinase 
Plt –                    Platelet 
RAS -                 Rat sarcoma 
SSC –                 Saline-sodium citrate  
STAT –               Signal transducer and activator of transcription proteins  
WBC -                White blood cell 
 
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ABSTRACT 
Background: Acute lymphoblastic leukaemia (ALL) is the accumulation of lymphoblasts in the 
bone marrow as a result of malignant transformation resulting in proliferation of immature 
lymphoid progenitors or lymphoblasts. It results from perturbation of several genetic loci. The 
chimeric BCR-ABL1 gene fusion is one of such genetic alterations. Even though the presence of 
BCR-ABL1 has been associated with poor prognosis, the incorporation of tyrosine kinase 
inhibitors in treatment protocols has been shown to be of enormous benefit. In Ghana as well as 
most African countries, research work on its prevalence and clinical associations is limited. 
Aim: To determine the frequency and associated laboratory and clinical features of the chimeric 
BCR-ABL1 gene in patients diagnosed with acute lymphoblastic leukaemia at the Department of 
Haematology, Korle Bu Teaching Hospital (KBTH). 
Methods: This is retrospective cross-sectional study. Methanol-fixed archived bone marrow 
aspirate slides of patients diagnosed with ALL at the Department of Haematology, KBTH were 
retrieved. Data on clinical features (signs) and haematological parameters was obtained from the 
patients’ medical records. The presence of the chimeric BCR-ABL1 fusion gene was determined 
on the bone marrow aspirate slides by fluorescent in situ hybridization (FISH).  
Results: A total of 17 cases were studied of which 13 (76.5%) were males and 4 (23.5%) were 
females. The ages of the participants ranged from 15 to 67 years ((mean =   31.5 years, SD = 
16.9 years).  A frequency of 29.4% was obtained for the BCR-ABL1 fusion gene. Of the clinical 
features studied, lymphadenopathy was present in 7 (40%) of study cases whereas splenomegaly 
and hepatomegaly were present in 4 (23.5%) and 5 (35.7%) respectively.  No significant 
association was established between BCR-ABL1 positivity and these clinical features. All 
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subjects had severe to moderate anaemia with haemoglobin concentration ranging from 3.7 to 
8.7g/dL. The mean haemoglobin concentration for BCR-ABL1 positive cases was higher than 
that of the negative cases (7.26 versus 6.62 g/dL respectively), however, statistical significance 
was not reached (P = 0.506).  The mean white blood cell count, bone marrow blast percentages 
and platelet counts were lower in BCR-ABL1 positive cases than in the negative cases (36.83, 
73.00 and 54.60 ×109/L versus 73.53, 82.18 and 74.33×109/L respectively) although no 
significant association was established between these haematological parameters and BCR-ABL1 
positivity (P = 0.879, 0.721 and 0.506 respectively).  Also, there was no statistically significant 
difference in clinical outcome between the BCR-ABL1 positive and negative cases. 
Conclusion: The BCR-ABL1 fusion gene is present in nearly one third of adult acute 
lymphoblastic leukaemia cases seen in this study and has no significant association with the 
clinical features and haematological parameters of the disease. A larger study will be needed to 
make a decision with regard to the modification of treatment regimen for adult BCR-ABL1 
positive ALL. 
 
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 CHAPTER ONE  
INTRODUCTION 
1.1 Background 
Acute lymphoblastic leukaemia (ALL) is the accumulation of lymphoblasts in the bone marrow 
as a result of malignant transformation resulting in proliferation of immature lymphoid 
progenitors or lymphoblasts (Ahmed, Dawson, Smith, & Wood, 2006). It  arises from several 
kinds of genetic alterations which affect haemopoietic  stem cells, early progenitor cells or genes 
that regulate the growth and differentiation of lymphoid cells (Nagarajan, 2010; Rose, 2013). 
Among these mutations is the ETV6-RUNX1 and hyperdiploidy which are generally associated 
with good prognosis and  hypodiploidy, MLL-AF4 and the BCR-ABL1 which are associated with 
poor prognosis (Mrozek, Harper, & Aplan, 2009). The BCR-ABL1  fusion gene results from 
translocations involving chromosomes 9 and 22 and gives rise to BCR-ABL1 positive ALL 
(Fainstein et al., 1987). Even though this mutation has been associated with poor prognosis, the
addition of tyrosine kinase inhibitors such as imatinib, nilotinib or dasatinib to treatment 
protocols has been shown to produce improved haematologic and cytogenetic remission rates  
(Ottmann et al., 2007). This study is aimed at obtaining the frequency and associated clinical 
features of the BCR-ABL1 fusion mutation among patients diagnosed with acute lymphoblastic 
leukaemia at the Korle Bu Teaching Hospital (KBTH). 
Acute lymphoblastic leukaemia has an estimated global incidence of 1 to 4.75 per 100, 000 
people (Redaelli, Laskin, Stephens, Botteman, & Pashos, 2005). Acute Lymphoblastic leukaemia 
accounts for about 20% of leukaemias in adults and 80% of childhood acute leukaemias making 
it the most common leukaemia in children. (Jabbour, O'Brien, Konopleva, & Kantarjian, 2015).  
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It has been shown to have the highest frequency of nearly 34% among all leukaemias diagnosed 
in the Korle Bu Teaching Hospital in Ghana (Ekem & Dei-Adomako, 2015).  A frequency of 
16.9%  was obtained in a study carried out in Nigeria (Damulak, Egesie, Jatau, Ogbenna, & 
Adediran, 2017). Furthermore, the findings of a study conducted at the Komfo Anokye Teaching 
Hospital in Ghana revealed that ALL accounted for 10% of  childhood cancers, the second most 
common after Burkitt’s Lymphoma (Painstil et al., 2015). 
 The chimeric BCR-ABL1 gene mutation occurs at varying frequencies in ALL in the range of 1-
5% and 11-29% in pediatric and adult cases respectively (Mrozek et al., 2009). The prevalence 
of BCR-ABL1 gene fusion in adult ALL cases from a multicenter study involving five cancer 
groups which includes the Cancer and Leukaemia group B and the MD Anderson Cancer Center  
is 20% whereas 15% was obtained in a population-based study of adult ALL cases in the United 
Kingdom (Anthony V Moorman, Chilton, et al., 2010; Roberts et al., 2015).In a study conducted 
in the South-western area of the Cape Province of South Africa, 9% of the patients diagnosed 
with ALL were blacks whereas 43% and 48% were of mixed ancestry (coloured) and white 
respectively (Jacobs, 1985). To date, no further studies have investigated the incidence of BCR-
ABL1 fusion in this population and many other regions in Africa resulting in a paucity of 
information.   
The cumulative mortality of 5-year survivors of childhood ALL at 25 years after diagnosis has 
been shown to be 13% in a cohort study conducted in United States and Canada.  (Mody et al., 
2008). The overall 5-year survival of newly diagnosed cases of adult ALL is 38% whereas 7% 
was observed for relapsed cases (Fielding et al., 2007).  
 
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Early allogenic bone marrow transplantation has been associated with improved overall survival 
in adult ALL cases particularly those associated with poor prognosis including those having the 
BCR-ABL1 gene fusion (75% 6-year overall survival compared with 41% for delayed autologous 
stem cell transplantations) (Hunault et al., 2004). Furthermore, the incorporation of tyrosine 
kinase inhibitors to therapeutic protocols has been associated with improved survival in BCR-
ABL1 positive ALL (Brissot et al., 2015; Fielding et al., 2014).  
 
1.2 Problem statement 
Acute lymphoblastic leukaemia is the accumulation of lymphoblasts in the bone marrow  
secondary to mutations in lymphoid stem cells (Ahmed et al., 2006; Hoffbrand & Moss, 2015). 
The chimeric BCR-ABL1 fusion gene which is the molecular equivalent of the Philadelphia 
chromosome results from translocation of the ABL cellular oncogene on chromosome 9 to the 
BCR gene on chromosome 22(Mullighan, 2012). This results in the synthesis of either a 210kD 
or 190kD protein with enhanced tyrosine kinase activity compared with the normal 145kD 
protein (Hoffman et al., 2012; Kumar, Abbas, Fausto, & Aster, 2009). 
The BCR-ABL1 fusion gene has been implicated as poor prognostic indicator in adult as well as 
childhood ALL as it has been associated with decreased overall and event free survival rates  
(Fletcher et al., 1991; Pullarkat, Slovak, Kopecky, Forman, & Appelbaum, 2008). Its frequency 
varies across different populations. Research on its frequency and clinical associations in Africa 
is limited and none has been carried out in Ghana. Since the prevalence of this mutant gene in 
ALL and its association with clinical features is unknown in Ghana, patients diagnosed with 
ALL are not routinely screened for its presence as it is costly. Treatment regimen is therefore not 
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adjusted for those who may be positive for the mutation even though tyrosine kinase inhibitors 
(TKIs) may be of enormous benefit to such patients and are available in Ghana.  
Acute lymphoblastic leukaemia has an estimated global incidence of 1 to 4.75 per 100, 000 
people (Redaelli et al., 2005).  The chimeric BCR-ABL1 gene mutation occurs at varying 
frequencies in ALL in the range of 1-5% and 11-29% in pediatric and adult cases respectively 
(Mrozek et al., 2009). 
In a study conducted in the South-western area of the Cape Province of South Africa, 9% of the 
patients diagnosed with ALL were blacks whereas 43% and 48% were of mixed ancestry and 
white respectively (Jacobs, 1985). To date, no further studies have investigated the incidence of 
BCR-ABL1 fusion in this population and many other regions in Africa. Therefore, the prevalence 
and the associated clinical signs and haematological parameters of this genetic alteration in 
patients diagnosed with acute lymphoblastic leukaemia in Ghana are not known. 
The overall 5-year survival of newly diagnosed cases of adult ALL is 38% whereas 7% was 
observed for relapsed cases (Fielding et al., 2007). The incorporation of tyrosine kinase 
inhibitors to therapeutic protocols has been associated with improved survival in BCR-ABL1 
positive ALL (Brissot et al., 2015; Fielding et al., 2014).  
1.3 Justification 
Optimum outcome is achieved if treatment modalities in ALL is tailored according to the 
cytogenetic and/or molecular genetic abnormalities present since they are associated with 
different prognosis (Cario et al., 2005; Anthony V Moorman, Ensor, et al., 2010; Slovak et al., 
2000). This study will provide information on the prevalence as well as associated clinical 
features and haematological parameters of the BCR-ABL1 fusion mutation among patients 
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diagnosed with ALL at the Department of Haematology, KBTH. The findings of this study may 
serve as the basis for informed decision making with regards to screening for BCR-ABL1 gene as 
part of the panel of tests for ALL patients and the establishment of the proper treatment protocol 
for the BCR-ABL1 positive cases thereby improving patient outcomes. 
 
1.4 Aim 
To determine the frequency and the associated laboratory and clinical features of the chimeric 
BCR-ABL1 gene in patients diagnosed with Acute lymphoblastic leukaemia at the Department of 
Haematology, Korle Bu Teaching Hospital (KBTH). 
 
1.5 Specific Objectives 
1. To determine the frequency of the chimeric BCR-ABL1 gene fusion in adult ALL 
patients seen at the Haematology Department, Korle Bu Teaching Hospital (KBTH). 
2. To determine the association between BCR – ABL1 positivity and clinical features of 
adult ALL patients. 
 
3. To determine the association between BCR – ABL 1 positivity and haematological 
parameters (white blood cell counts, platelet counts, haemoglobin concentrations and 
bone marrow blast percentage) of adult ALL patients. 
 
4. To determine association between BCR-ABL1 positivity and treatment outcome of adult 
ALL patients. 
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CHAPTER TWO  
LITERATURE REVIEW 
2.1 Definitions  
Acute lymphoblastic leukaemia (ALL) is the accumulation of lymphoblasts in the bone marrow  
secondary to mutations in lymphoid stem cells (Ahmed et al., 2006; Hoffbrand & Moss, 2015). It 
is characterized by the  presence of more than 20% leukaemic blasts in the bone marrow at 
clinical presentation although a lesser percentage is also definitive if specific leukaemic 
cytogenetic or molecular genetic abnormalities are present (Hoffbrand & Moss, 2015). 
Among the genetic mutations implicated in the development of ALL is the chimeric BCR-ABL1 
fusion  gene which is the  molecular equivalent of the Philadelphia chromosome which results 
from translocation of the ABL  cellular oncogene on chromosome 9 to the BCR gene on 
chromosome 22 (Shtivelman, Lifshitz, Gale, & Canaani, 1985). This results in the synthesis of 
either a 190kD  or 210kD protein with enhanced tyrosine kinase activity compared with the 
normal 145kD protein (Hoffman et al., 2012; Kumar et al., 2009).  The p190 is the more 
common in ALL than the p210. The frequency of the p190 is in the region  50 to 70%  and 80% 
for adult and pediatric cases respectively may occur simultaneously with the p210 in up to 19% 
of BCR-ABL1 positive ALL(Hoffman et al., 2013). The incidence of the BCR-ABL1 gene has 
been shown to vary across different ethnic groups in a study conducted in childhood ALL 
(Ariffin et al., 2007).  
 
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2.2 Classification 
Acute lymphoblastic leukaemia may be either of a B-cell or a T-cell lineage. About 85% of the 
cases are B cell with equal incidence in both sexes whereas the remaining 15% has male 
predominance and originate from the T cell.  (Hoffbrand & Moss, 2015)  The table below shows 
the detailed immunophenotypic classification. The determination of 
 the immunophenotype usually by flow cytometry aids in the establishment of diagnosis, 
monitoring response to therapy and prognostic assessment  (Alves et al., 2012).   
Table 1: IMMUNOPHENOTYPIC CLASSIFICATION OF ACUTE LYMPHOBLASTIC 
LEUKAEMIA 
 Early Pre- Mature Pro-T Pre-T Cortical Mature REFERENCES 
Pre-B B B T T 
TdT + + - + + + +/- 1.(Abeloff, 
HLA- + + + +/- - - - Armitage, 
DR Niederhuber, Kastan, 
CD10 - +/- +/- +/- +/- +/- - & McKenna, 2008) 
CD19 + + + - - - -  
CD22 + + + - - - - 2. (Cairo & Perkins, 
CD79a + + + - - - - 2012) 
cIgM - + - - - - - 
sIg  - - + - - - - 
cCD3 - - - + + +/- - 
mCD3 - - - - - +/- + 
Ccd7 - - - + + + + 
CD2 - - - - + + + 
CD1a - - - - - + + 
 
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Some myeloid associated cluster of differentiation (CD) antigens are aberrantly expressed in 
ALL especially in BCR-ABL1 positive cases.  These markers which include CD13, CD33, CD14 
and CD15 are good indicators of the presence of leukaemic blasts and minimal residual disease 
although they have no prognostic importance (Stefan Faderl et al., 2010). 
Acute lymphoblastic leukaemia may be classified as BCR-ABL1 positive or negative (the 
molecular counterpart of Philadelphia positive or negative ALL) based on the results of BCR-
ABL1 testing whether positive or negative respectively and this is of prognostic significance  
(Nashed, Rao, & Gulley, 2003; Terwilliger & Abdul-Hay, 2017).  
 
2.3 Epidemiology        
Acute Lymphoblastic leukaemia occurs in both pediatric and adult populations with peak 
incidence at the age of 2 to 5 years (Hiroto Inaba, Mel Greaves, & Charles G. Mullighan, 2013; 
Levy, 2010). The second peak incidence occurs in adults over 50 years (Lysaght et al., 2013). It 
has an estimated global incidence of 1 to 4.75 per 100, 000 people (Redaelli et al., 2005). It has 
been shown to have the highest frequency of nearly 34% among all leukaemias diagnosed in the 
Korle Bu Teaching Hospital in Ghana (Ekem & Dei-Adomako, 2015) . About 60,000 new cases 
are recorded annually in the United States of America with a male to female ratio of 1.3:1 
(Hiroto Inaba et al., 2013).  
The chimeric BCR-ABL1 gene mutation occurs at varying frequencies in ALL in the range of 1-
5% and 11-29% in pediatric and adult cases respectively (Mrozek et al., 2009). A frequency of 
42% to 44% beyond age 44 has been reported in Germany (T. Burmeister et al., 2008).  It was 
detected in 22% of the 18 cases of childhood ALL that were studied in Sudan (Siddiqui et al., 
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2010).  Also, 12.5%  of the 40 cases of childhood and adult ALL cases in a study conducted in 
Nigeria were BCR-ABL1 positive (Ajuba et al., 2016). A prevalence of 28.3% was recorded in 
one study in India (Chopra et al., 2015). 
In a study carried out in Saudi Arabia, none of the 16 cases of adult ALL investigated was 
positive for the BCR-ABL1 fusion gene (El-Sissy, El-Mashari, Bassuni, & EL-SWAAYED, 
2006).  
 In one study, no significant association was found to exist between BCR-ABL1 positivity and 
age as well as gender (Hamid & Bokharaei, 2017) 
The two main types of the BCR-ABL1 fusion transcripts (p190 and p210) occur in ALL but the 
p190 is more prevalent (Cimino et al., 2006; Gleißner et al., 2002). In a study of 56 adult ALL 
cases in the United States of America by the Cancer and Leukaemia Group B (CALGB), the 
p190 variant accounted for 77% of the cases whereas the p210 was detected in the remaining 
23% (Westbrook et al., 1992). 
 
2.4 Aetiology 
The acute lymphoblastic leukaemias arise from genetic mutations in the haemopoietic cells or 
early progenitor cells (Rose, 2013). The mutations result in malignant transformations of these 
cells by three mechanisms which include the following: 
I) Enhanced rate of self-renewal and proliferation; 
II) Impaired apoptosis resulting in survival advantage and   
III) arrest of cellular differentiation (Hoffbrand & Moss, 2015). 
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These events result in overproduction of lymphoblasts which accumulate in the bone marrow and 
replace the normal cells as well as spill over into blood and infiltrate organs (Rose, 2013). 
Also, mutations in the genes that regulate the growth and differentiation of lymphoid cells 
resulting in abnormal production of their respective proteins have also been implicated in the 
pathogenesis of acute lymphoblastic leukaemia (Nagarajan, 2010). 
 
2.5 Cytogenetics and Molecular genetics 
Some of the genetic alterations found in ALL include altered chromosome number (hyerdiploidy 
and hypodiploidy), translocations which include t(12,21), t(4,11) and t(9,22) which give rise to 
ETV6-RUNX1  , MLL-AF4 and the BCR-ABL1 fusion genes respectively (Mrozek et al., 2009). 
In order for ALL to be manifested, these chromosomal abnormalities often act in tandem with 
some other genetic lesions which include deletions or silencing of CDKN2A gene, alterations in 
the PAX 5 gene and deletional changes affecting the E2A, EBF1 and IKZF family genes (Pui, 
Robison, & Look, 2008). Of these, PAX 5 is the most common mutated gene and occurs in 
nearly 32% of cases (Mullighan et al., 2007) 
IKZF1 gene deletion mutations have been shown to play major contributory role in the 
development of BCR-ABL1 positive acute lymphoblastic leukaemia (I. Iacobucci et al., 2009). 
Also, PAX 5, CDKN2A/ARF and CDKN2B deletions have been associated with BCR-ABL1 
positive ALL (Iacobucci et al., 2011). 
The exact causes of the genetic mutation in most cases of ALL are not well elucidated; however, 
certain inherited and environment factors have been implicated in the aetiogenesis of these 
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events as they have been associated with increased risk of development of ALL and are 
discussed below (Chang et al., 2007).  
In a higher proportion of cases, the disease arises spontaneously in otherwise healthy individuals 
without any predisposing factors (Terwilliger & Abdul-Hay, 2017) 
 
2.6 Risk Factors 
The risk factors are both inherited and environmental and include the following: 
2.61 Inherited factors 
The inherited genetic disorders that increases the predisposition to the development of ALL are 
Down’s  syndrome, Klinefelter’s syndrome, Fanconi’s anaemia, Bloom’s syndrome and ataxia-
telangiectasia (Chang et al., 2007; Pui et al., 2008). These risk factors are associated with only a 
minority of cases (less than 5%) except in children with Down’s syndrome in which the risk is 
increased to about 40-fold below age five (H. Inaba, M. Greaves, & C. G. Mullighan, 2013; Paul, 
Kantarjian, & Jabbour, 2016). 
Even though ALL in Down’s syndrome (trisomy 21) has not been associated with any 
cytogenetic abnormality, the over-expression of proto-oncogenes due to the extra 21st 
chromosome may account for the leukaemic transformations (Chang et al., 2007; Harris, 2015).  
Fanconi’s anaemia, Bloom’s syndrome and ataxia-telangiectasia are associated with increased 
chromosomal instability which increases the risk of  developing ALL (Chang et al., 2007). 
The initiating mutation events (first hit) in childhood ALL occur in utero or early infancy during 
which lymphocyte expansion and recombinase activity are climactic whereas the promotional 
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mutation (second hit) which occurs afterwards and result in clinical manifestation of ALL is as a 
result of exaggerated response of the immune system to exogenous antigens resulting in over-
proliferation of lymphoid cells (Bope & Kellerman, 2011). 
2.62 Environmental risk factors 
Exposure to certain drugs such as ALLN (N-acetyl-Leu-Leu-Norleu-al ), alkylating agents  and 
topoisomerase II inhibitors such as etoposide and epipodophyllotoxins have been associated with 
increased risk of ALL development (Appelbaum, Forman, Negrin, & Blume, 2011). 
Chemicals such as benzene, pesticides, automobile exhaust , parental alcohol consumption and 
cigarette smoking have been proposed as etiologic risk factors and are being investigated (Estey, 
Faderl, & Kantarjian, 2007). 
Also, in utero and post-natal  exposure to radiation from atomic bombs (atomic bomb survivors 
of Hiroshima and Nagasaki), nuclear explosions and medical treatment  has been implicated in 
the aetiology of ALL (Cullings, 2014; Weiner & Cairo, 2002). Even though diagnostic X-rays 
have not been associated with ALL development, intrauterine foetal exposures have been linked 
with increased risk of ALL in childhood (Vokes & Golomb, 2011). Low-frequency 
electromagnetic field has not been shown to pose any risk (Weiner & Cairo, 2002). 
Even though no specific infectious agent has been implicated in the aetiogenesis of ALL, the  
second hit mutations resulting from abnormal immune response to infectious agents in infants 
who were not exposed to common infections in the first years of life has been linked with 
increased risk of ALL (Hayat, 2013; Pui et al., 2008). 
 
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Increased birth weight and advanced maternal age  are have also been suggested as risk factors 
but with little evidence (Weiner & Cairo, 2002). Neonatal vitamin K administration and 
increased maternal intake of diets rich in nitrites have also been proposed (Pui & Evans, 1998).  
Even though an association was found between maternal exposure to second-hand tobacco 
smoking during pregnancy as well as childhood exposures and ALL, none was observed for 
maternal smoking and paternal smoking during pregnancy (Farioli et al., 2014). 
 
2.7 Pathogenesis 
The exact pathogenetic mechanism by which ALL occurs is not known as only a minority of 
cases have been directly linked to risk factors. Among the proposed mechanisms include a 
pathological exaggerated immune response after exposure of non-immune individuals with 
prenatal leukaemia cell lines to common environmental pathogens; influenza viruses have been 
implicated in childhood ALL (Greaves, 2018; Kroll, Draper, Stiller, & Murphy, 2006).  
Polymorphism and alterations of genes involved in some metabolic and cell-signalling pathways 
are believed to play a role in ALL pathogenesis. The polymorphic expression of the GST T1 null 
variant of the glutathione S- transferase family of genes has been associated with increased 
development of adult ALL (Rollinson et al., 2000) . This most likely results from DNA damage 
arising from decreased detoxification of carcinogens and removal of reactive oxygen species 
which are key functions of the gene (Singh & Michael, 2009).  
The MTR 2756GG variant of the methionine synthase gene (MTR 2756 A>G) involved in the 
transfer of single carbon atoms in DNA methylation has been associated with the development of 
acute lymphoblastic leukaemia (Lightfoot et al., 2010).  
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Genetic alterations resulting in enhanced kinase signalling such as those involving the 
PI3K/AKT/mTOR,  IL-7R/JAK/STAT and RAS/MAPK pathways have been associated with the 
leukaemogenesis of T-ALL  (Bongiovanni, Saccomani, & Piovan, 2017). 
 
2.8 Clinical features and laboratory findings 
 Clinical features include anaemia, thrombocytopenia and neutropenia which is as a result of 
bone marrow failure, as well as organ infiltration which manifests as lymphadenopathy, 
moderate splenomegaly, hepatomegaly and meningeal syndrome (Ferri, 2015). Mediastinal mass 
also occurs in ALL but has been associated with T-lineage rather than  B-lineage ALL (Rossi et 
al., 1993). Constitutional symptoms such as fever, weight loss and night sweats as well as easy 
bruising,, dyspnoea, fatigue and infection emanating from decreased blood cell counts are the 
most prevalent symptoms in ALL (Terwilliger & Abdul-Hay, 2017). 
In a study conducted in Egypt, lymphadenopathy, splenomegaly and hepatomegaly occurred at 
frequencies of 58, 58 and 54% respectively in adult ALL (Elbossaty, 2017). However, a 
combined frequency of 40% was obtained in another study of Adult ALL in Netherlands 
(Daenen et al., 1998).  
BCR-ABL1 positive ALL cases have been linked with higher white cell count and haemoglobin 
levels as compared to BCR-ABL1 negative cases. There is a similar incidence of splenomegaly, 
lymphadenopathy, mediastinal mass and hepatomegaly (Gleißner et al., 2002; Westbrook et al., 
1992). However, no statistically significant difference was established between haemoglobin and 
white blood cell counts of BCR-ABL1 positive and negative cases (Westbrook et al., 1992).  
Also, lower platelet counts (P=0.07) and higher peripheral blood blast  counts (P=0.02) have 
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been linked with the BCR-ABL1 positive ALL compared with the negative cases (Pullarkat et al., 
2008). 
 
2.9 Laboratory Diagnosis  
The initial laboratory tests to be carried out include  complete blood count and  examination of 
Romanowsky stained peripheral blood and bone marrow aspirate slides for the presence of 
increased numbers of lymphoblasts exceeding 20% of nucleated cells (Theml & Diem, 2011). 
The lineage of the lymphoblasts as well as stage of maturation is confirmed by 
immunophenotyping usually by flow cytometric detection for the expression of cluster of 
differentiation marker antigens. The use of cytochemical stains such as periodic acid Schiff 
reagent may be used to different ALL from acute myeloid leukaemia (Sun, 2012).  
The next stage of diagnosis involves karyotyping in which chromosomes are examined for gross 
genetic changes such as deletions and translocations which include the t(9,22) (Ludwig & Thiel, 
2012). The determination of the specific genetic lesions involved in these gross chromosomal 
changes such as the BCR-ABL1 gene for t(9,22) may be carried out using molecular cytogenetic 
tests such as fluorescent in situ hybridisation or molecular genetic tests such as next generation 
sequencing, microarray analysis and polymerase chain reaction-based techniques (Leonard, 
2016).  
Typical fluorescent in situ hybridisation signal patterns for detection of BCR-ABL1 fusion gene 
include 2 fusion signals, 1 red and 1green,  whereas one fusion signal is observed in deletions 
involving both the BCR and ABL genes; normal signals appear as 2 red and 2 green dots (Jain et 
al., 2012). 
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2.10 Treatment 
The treatment of ALL is achieved with chemotherapy involving mainly 4 stages namely  
induction, consolidation, central nervous system prophylaxis and maintenance therapy (S. Faderl 
& Kantarjian, 2011). The table below shows the various stages and the drugs that may be used. 
Table 2: DRUGS USED IN THE TREATMENT OF ACUTE LYMPHOBLASTIC LEUKAEMIA  
Remission  Consolidation CNS Maintenance References 
Induction Prophylaxis 
vincristine Daunorubicin  Mercaptopurine (Hoffbrand & 
anthracycline Cytarabine Intrathecal Vincristine Moss, 2015). 
  Vincristine methotrexate methotrexate (Florin, Ludwig, 
asparaginase and Etoposide  Aronson, & 
cyclophosphamide mitoxantrone Werner, 2011) 
(optional)  
 
The drugs in the table above are used in various combinations and cycles with age, gender and 
leucocyte counts at the onset of the disease being key determinants of treatment modalities.  
Treatment may last between 2 and 3 years and the intensity of the regimen, duration  and 
whether or not stem cell transplantation will be carried out depends on the specific subtype and 
the risk of relapse (Kaye` & Kaye, 2004).  High intensity regimen are used for pediatric and 
young adults as well as high risk factors such as presenting white blood cell counts exceeding  50 
× 109/uL whereas low doses are used for older adults (Rabin & Poplack, 2011). 
The addition of tyrosine kinase inhibitors such as imatinib to treatment protocols in BCR-ABL1 
positive ALL has been associated with increased overall and disease-free survival rates (Bassan 
et al., 2010; Schultz et al., 2009).  
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2.11 Prognosis 
The prognosis of ALL is poorer in adults than children and adolescents with 5-year survival rates 
of 40% and 90% respectively  (Hunger et al., 2012; Pulte et al., 2014) 
Even though BCR-ABL1 positive and negative cases of ALL have been shown to have similar 
remission rates, more early relapses have been associated with BCR-ABL1 positive ALL 
(Westbrook et al., 1992). Secondly, in a Cancer and Leukaemia Group B study in the United 
States of America, the median survival duration in BCR-ABL1 positive and negative ALL was 
11.2 and 21.8 months respectively but was statistically insignificant (p-value 0.26) (Westbrook et 
al., 1992). In a multicenter study conducted in Germany, the 3-year survival probability in BCR-
ABL1 positive versus negative ALL was 0.19 (+/-0.04SE) and 0.55(+/-0.04SE) respectively (p-
0.0001) (Gleißner et al., 2002). Also, the BCR-ABL1 positive ALL has been significantly 
associated with decreased overall and 5-year event-free survival compared to negative cases (A. 
V. Moorman et al., 2007). However, in an Italian based study,  the  expression of the  PAX5 
wild-type without IKZF1 deletion in BCR-ABL1 positive ALL  has been associated  with good 
prognosis (prolonged disease-free survival and low incidence of relapse) compared with cases 
expressing normal PAX5 and 1KZF1 deletion (Ilaria Iacobucci et al., 2009).  
In a study conducted in Nigeria, no association was found between BCR-ABL1 positivity and 
white cell count  (p = 0.416) )  (Ajuba et al., 2016) even though higher WBC count is associated 
with ALL and considered a poor prognostic factor (Advani & Lazarus, 2010; S. Faderl & 
Kantarjian, 2011) . In a south African based study of a heterogenous ethnic pediatric population, 
no significant statistical association was found between 5-year event free survival rate and 
clinical features (hepatomegaly, splenomegaly and mediastinal mass) as well as 
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immunophenotype (p = 0.9, 0.38, 0.41 and 0.87 respectively) (Wessels, Hesseling, Buurman, 
Oud, & Nel, 1997). 
 
2.12 Monitoring 
The detection of BCR-ABL1 rearrangement has been proven to provide significant guidance in 
the diagnosis, prognosis, monitoring of response to treatment and drug resistance in acute 
lymphoblastic leukaemia (Jiang et al., 2016). The detection of BCR-ABL1 transcripts by the 
polymerase chain reaction technique has been proven to be useful in the detection of minimal 
residual disease in Philadelphia-positive ALL as it aids in the monitoring of the effect of 
treatment and detection of relapse (Miyamura et al., 1992).  
 
 
 
 
 
 
 
 
 
 
  
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CHAPTER 3  
METHODOLOGY 
 
3.1 Study design 
This is a retrospective cross-sectional study in which methanol-fixed archived bone marrow 
slides of patients diagnosed with ALL were used. 
3.2 Study site 
The study was carried out at the Department of Haematology, Korle Bu Teaching Hospital.  The 
hospital  is the third largest hospital in Africa and the leading referral centre in Ghana 
(https://kbth.gov.gh/korle-bu-trust-fund.html, 2016).The Department of Haematology provides 
laboratory and clinical services for patients with various haematological disorders from all over 
Ghana  as well as neighbouring West African countries. About 4800 patients are seen at the 
department each year. In the hospital, an average 28 cases of ALL are diagnosed each year with 
12 of them being adults. 
The bench work of the project was carried out by the investigator at the Queen’s Laboratory for 
Molecular Pathology at Queen’s University in Canada where training in the fluorescent-in situ 
hybridisation technique was acquired and the bench work carried out within a three-month 
period. This trip was necessitated by the fact that the FISH procedure could hardly be carried out 
in Ghana due to unavailability of necessary equipment and technical expertise. 
 
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3.3 Study Population 
The population consists of diagnosed cases of ALL at the Department of Haematology, KBTH 
from January 2013 to MAY 2017. 
 
3.4 Inclusion criteria 
1. Study cases must be patients ≥15 years old. 
2. Cases should have been morphologically diagnosed as ALL. 
3. The unstained bone marrow aspirate slides of cases should be available. 
3.5 Exclusion criteria 
1. Cases whose archived bone marrow aspirate slides and/or laboratory report at diagnosis 
were not available were excluded.  
2. Cases with incomplete data from folders were excluded. 
3.6 Sample size determination  
The sample size was determined using the Cochran’s sample size formula which is given by the 
following equation: 
                 n = Z 2p(1 – p)     
d2 
Where  
n = minimum sample size 
Z is Z score (Z=1.96 for confidence level of 95%) 
Prevalence of BCR-ABL1 fusion gene in ALL = 11 – 29%....... (Mrozek et al., 2009) 
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        Using the minimum value of the prevalence range ⇒ p = expected proportion= 11%  
           d = accepted margin of error = 5% 
1.96×1.96×0.11(1 – 0.11)
Therefore n =  = 150.43 
0.05×0.05
  
But the study is retrospective cross-sectional (January 2013 to December 2015) with a finite 
population made up of a total of 37 cases. 
This finite population is corrected using the formula below:  
         n’= n ×N)/[n +(N – 1)] ……………………………….(Naing, Winn, & Rusli, 2006) 
where n’= corrected sample size for a finite population 
            N = finite population size = 37 
            n = sample size without taking the finite population correction into consideration = 151 
        ∴ n’ = 151 × 35/ [151 + (37 – 1)] = 28.6 ≈ 30. 
However, only 25 of the 37 cases seen in the study period had archived bone marrow aspirate 
slides available.  Therefore, even though 30 is obtained from the sample size calculation above, 
the sample size used for the study was 25. Hence 25 bone marrow aspirate slides were selected 
for the study. 
3.7 Selection of samples and data collection 
Unstained bone marrow aspirate slides of study cases (adults ALL) were retrieved from storage 
using laboratory numbers obtained from the Haematology laboratory log book. The storage 
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comprised of all bone marrow aspirate slides of all haematological disorders prepared from 
January 2013 to May 2017. 
The data abstraction form shown on appendix A was used to obtain the following information  
from the patients’ medical records: 
i) Clinical features present at presentation (i.e. hepatomegaly, splenomegaly, 
lymphadenopathy and/or presence of mediastinal mass) 
ii) Laboratory variables were obtained from FBC which was performed at the time bone 
marrow aspirate was taken for diagnosis (i.e. haemoglobin concentration, white cell and 
platelet counts). The blast percentages were obtained from bone marrow aspirate smears. 
 
3.8 Materials and Methods 
The fluorescence in-situ hybridisation (FISH) technique was used for the detection of the BCR-
ABL1 fusion gene in the unstained archived bone marrow aspirate slides. 
Equipment and apparatus used include the following: 
1. ThermoBrite Denaturation/Hybridisation System (Abbot Molecular) 
2. Fluorescent Microscope (OLYMPUS BX61) 
3. Computer with imaging software - GENASIS FISHView and Case Data Manager (CDM) 
(from Applied Spectral Imaging) 
4. Phase Contrast Microscope (Zeiss Axio Lab. A1) 
5. Water bath 
6. Microcentrifuge  
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7. Vortex Mixer 
8. Digital Thermometer 
9. Micropipettes 
 
The reagents used are listed in appendix C. 
 
3.9 Procedure 
The fluorescent in-situ hybridisation technique was performed by the investigator using the 
protocol of the Queens Laboratory for Molecular Pathology (Queens University, Canada):   
procedure as described below: 
Preparation of Positive and Negative Control Smears 
A positive control smear was prepared from a cell culture of a commercially prepared BCR-   
ABL1 positive cell line [K-562 (ATCC ® CCL -243)].  A peripheral blood smear prepared from a 
BCR-ABL1 negative anonymised subject which was provided by the Queens Laboratory for 
Molecular Pathology was used as a negative control slide. Both slides were fixed in methanol for 
3 minutes. 
The FISH procedure was carried out on study cases and control slides as per the protocol of the 
Queens laboratory for Molecular pathology which is as follows: 
 
 
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DNA Unmasking 
The slides were immersed in methanol for 1 minute followed by incubation in 2× SSC at room 
temperature for 5 minutes.  
The slides were then incubated in 0.2N HCl for 5minutes. 500ul of pepsin was added to the 
prewarmed 49.5ml of 0.01N HCl in water bath at 37oC. The resulting mixture was mixed well. 
The slides were taken from the 0.2N HCl solution and excess liquid remove with paper towel 
and immediately immersed into the pepsin/HCl solution incubating at 37oC for 7 to 15 minutes. 
The Slides were washed in ddH2O for 10minutes followed by fixation in 1% formaldehyde for 5 
minutes, immersion in 1% phosphate buffered saline for 5 minutes and sequential dehydration in 
70%, 85% and 100% ethanol for 2 minutes in each solution. 
The slides were observed under phase contrast microscope for digestion progress (Unfinished 
digestion is signified by the appearance of white shiny cells with undefined nuclei whereas cells 
with clear blue nuclei shows complete digestion). If digestion is not finished, the slides were re-
immersed in pepsin/HCl solution for a longer period and the subsequent steps followed until the 
phase contrast microscope shows a finished digestion. 
 
Denaturation and Hybridization 
The slides were air dried completely prior to the addition of the probe in the hybridisation steps 
which is described in the following paragraphs: 
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The ThermoBrite Denaturation/Hybridisation system was turned on and the program set for the 
following parameters:                                                                                                          
Denaturation time: 2 mins;                  Denaturation   temperature: 73oC ;                          
Hybridisation Time: 24 hrs;                 Hybridisation temperature: 37oC  . 
The DNA probe (Vysis LSI   BCR/ABL DC/DF translocation probe), Vysis LSI/WCP 
hybridisation buffer and the purified water were removed from storage and the reagents allowed 
to reach room temperature. The BCR/ABL DNA probe and the hybridisation buffer were 
vortexed for 2 to 3 seconds followed by centrifugation for 2 to 3 seconds. 
Seven microlitres (7uL) of the hybridisation buffer, 2uL of purified water and 1uL of the 
BCR/ABL DNA probe were transferred into a microcentrifuge. The mixture was vortexed and 
centrifuged for 2 to 3 seconds each. 
A micropipette was used to apply 10uL of the probe mixture to the target area of each slide. A 
cover slip was immediately applied without introducing bubbles. The coverslip was sealed using 
a syringe filled with rubber cement. 
Two ThermoBrite humidity cards saturated with distilled water were inserted into the slot 
positions in the unit lid of the ThermoBrite Hybridisation/Denaturation system.  
The slides were placed on the heating surface of the ThermoBrite Hybridisation/Denaturation 
system when prompted and was ensured that, the frosted edge of the slide hanged over the 
heating surface, lay flat and properly aligned into the marked positions in the slide locator.  The 
ThermoBrite lid was closed and the program was started for denaturation and hybridisation to 
occur overnight. 
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Post-Hybridisation Wash 
The post-hybridisation wash was carried out according to the following procedure: 
The room was darkened and the coverslip removed from the slide by peeling off rubber cement. 
The slides were incubated in 2× SSC/0.3% IgePal solution (180uL IgePal in 60ml 2× SSC) at 
73oC for 2 minutes. The temperature was increased by 0.5oC for each slide if the slides were 
more than one. The slides were washed in 2× SSC for 5minutes and air-dried in upright position 
under foil cap. 
 
Counterstaining  
Counterstaining was carried out by the application of 10uL DAPI to the middle of each slide. 
Coverslip was applied and air bubbles pushed out. They were stored in the dark at -20oC until 
fluorescent microscopy was carried out in the following step. 
Fluorescent Microscopy 
The fluorescent microscopy room was darkened and immersion oil added to the slides. They 
were then observed under the fluorescent microscope using the spectrum orange, spectrum green 
and the dual filter which allows the visualisation of ABL1, BCR and BCR/ABL1 gene 
respectively. A total of 100 interphase nuclei were scored for each slide. Images of the slides 
were captured using the imaging software GENASIS FISH View and processed using Case Data 
Manger (CDM). Representative images have been shown in section 4.2. 
 
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3.10 Data Handling 
Study cases were assigned unique identification numbers. It is these identification numbers that 
were used to label the respective bone marrow aspirate slides and also for subsequent data 
processing.  
The data was stored on a password protected computer. The names of subjects were not used in 
the data but were however kept in a different file.  Only the Investigator and his supervisors had 
access to data obtained from the study. 
 
3.11 Statistical analysis                             
The data was entered into Microsoft Excel and exported to Statistical Package for the Social  
Sciences (SPSS) for analysis. Data was expressed using summary and descriptive statistics such 
as frequency, percentages and median as appropriate and presented in tabular form. Chi Square, 
Fisher exact test and non-parametric test (Mann-Whitney U-test) were used to assess the 
association between categorical factors. 
 
 
 
 
 
 
 
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CHAPTER 4  
RESULTS 
A total of 25 bone marrow aspirate slides were selected for the study. However, FISH was 
successfully carried out on 17 as 8 slides were not suitable to be used for the test. Four of the slides 
upon examination under the microscope had structures suspected to be fungal hyphae covering the 
entire slide thus they could not be used for analysis. The remainder could not withstand experiment 
procedures as the glass slides used to prepare the bone marrow aspirate smears were not 
electrostatically charged type as such the cells were lost during the experimental procedure. 
Therefore, only slides from 17 patients were available for analysis as shown in figure 1. 
 
 
 
 
 
 
 
 
 
 
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 Retrieval of Marrow Aspirate Slides of Haematological Cases 
from Storage (≈ 1600 patients’s slides) 
                                    
              
                                                                              ≈ 
                                                   
 
 Selection of Adult ALL Slides (52 SLIDES).  Study population 
 
includes only adult ALL cases. Pediatrics ALL and all cases other 
                                                     than ALL were not included. .  
 
                                                                                                                                            
 Selection of Adult ALL Slides with Folders and Laboratory Report 
Available (25 Slides). Post-treatment ALL slides and slides with 
                            n  o    f  olders and laboratory report available were not included. . 
 
 
 
                                                                                     
Selection of Adult ALL Slides Based on Quality for FISH 
                              A   n  alysis(17 Slides).  Slides with no observable haemopoietic 
cells prior to FISH experiment and slides in which cells were lost 
 during experiment were rejected. 
 
                   Figure 1: FLOW DIAGRAM FOR THE SELECTION OF BONE MARROW 
ASPIRATE SLIDES FOR THE STUDY 
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4.1 Fluorescence In Situ Hybridization results and frequency of the BCR-ABL1 gene in 
samples. 
The table below shows the results of detection of the presence of BCR-ABL1 fusion gene on 
unstained bone marrow aspirate slides of study cases by fluorescent in situ hybridization.  
  
Table 3: FISH RESULTS FOR BCR-ABL1 FUSION GENE 
 
BCR-ABL1 gene 
results Frequency Percent 
 
Negative 12 70.6 
Positive 5 29.4 
Total 17 100.0 
 
From the table above, it can be seen that, 29.4% (5 cases) were positive for the BCR-ABL1 gene. 
Therefore, the frequency of the BCR-ABL1 fusion gene for the adult Acute Lymphoblastic 
Leukaemia samples tested in this study was 29.4%.  
The cut-off point for positivity was 1% and 15% for double fusion and single fusion respectively. 
Of the 5 positive cases, 4 were double fusion and one had single fusion. 
The percentage scores for positive cases of the gene were 46%, 37%, 9% and 8% for double 
fusion and 20% for single fusion. 
 
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4.2 Selected FISH Images 
The figure below shows a fluorescent photomicrograph of the negative control slide. No fusion 
signals are present. 
 
 
Figure 2: Negative Control 
 
 
 
 
 
 
 
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The figure below shows the fluorescent photomicrograph of a study participant with no fusion 
signals present (negative case) 
  
Figure 3: BCR-ABL1 fusion Negative Case 
 
 
 
 
 
 
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The figure below shows the fluorescent photomicrograph the BCR-ABL1 positive control smear. 
Double fusion signals which appear yellow are seen in the lower left and topmost nuclei. 
 
 
Figure 4: Positive Control 
 
 
 
 
 
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The figure below shows a fluorescent photomicrograph of a BCR-ABL1 positive case. Double 
fusion signals (either yellow dots or orange and green dots in juxtaposition) are seen in the two 
nuclei in the middle. 
 
Figure 5:BCR-ABL1 fusion Positive Case 
 
 
 
 
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In the figure below, a single fusion signal (yellow) is seen in the larger cell. 
 
Figure 6: BCR-ABL1 fusion case showing a single fusion signal 
 
KEY TO INTERPRETATION OF IMAGES 
BCR-GENE – Green signals (dots) 
ABL GENE – Orange signals (dots) 
BCR-ABL1 FUSION GENE – Orange and green signals in juxtaposition or yellow signal are 
seen for BCR-ABL1 positive cases. Double fusion cases have 2 signals whereas 1 signal is seen 
single fusion cases). BCR-ABL negative cases show separated green and orange signals but no 
fusion signals).  
 
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4.3 Demographics                                                        
Table 4: BCR-ABL1 FUSION GENE AND SEX 
BCR-ABL1 P-value 
RESULTS (Fisher 
 
exact 
 Negative Positive Total test) 
SEX female Frequency 3 1 4  
% within  
75.0% 25.0% 100.0% 
SEX 0.67 
male Frequency 9 4 13 
% within 
69.2% 30.8% 100.0% 
SEX 
Total Frequency 12 5 17 
% within 
70.6% 29.4% 100.0% 
SEX 
 
In the study 76.5% (13)  of the samples were drawn from males and 23.5% (4) were from 
females. From the table above, the proportion of BCR-ABL1 positive cases was lower compared 
to BCR-ABL1 negative cases (25.0% versus 75.0% within the female category and 30.8% versus 
69.2% within the male category). From the Chi-Square test, there is a likelihood of no significant 
association between BCR-ABL1 positivity and sex of study cases. (p-value = 0.67). 
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4.4 Age 
Participants in the study were categorized into two groups; adolescent and young adult group 
(AYA group) and older adult group with the age ranges from 15 to 39 years and 40 years and 
above respectively based on the guidelines of the National Comprehensive Cancer Network 
(NCCN), U.S.A. The corresponding mean ages were 22.2 (+/-7.7) years and 53.8 (+/-10.0) years 
respectively.  
The ages of participants ranged from 15 years to 67 years (mean =   31.5 +/- 16.9 years). Table 5 
shows the proportion of each group. 
Table 5: BCR-ABL1 FUSION GENE AND AGE 
  
BCR-ABL1 P-value 
RESULTS (Fisher 
exact 
 Negative Positive Total test) 
Age AYA Frequency 7 5 12  
Group % within  
58.3% 41.7% 100.0% 
AYA group 0.245 
Older Frequency 5 0 5 
Adults % within 
Older Adult 100.0% 0.0% 100.0% 
group 
Total Frequency 12 5 17 
% within both 
AYA Older 70.6% 29.4% 100.0% 
Adult group. 
 
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In the table above, BCR-ABL1 positive cases constitute a lower proportion than BCR-ABL1 
negative cases within each age group (41.7% versus 58.3% in the AYA group and 0.0% versus 
100.0% in the Older Adult group.  The p-value is = 0.245, hence there is no significant 
association between the BCR-ABL1 gene positivity and age groups (adolescent and young adult 
group and older adults.                                                                                                     There 
were 12 cases (70.6%) in the AYA group and 5 cases (29.4%) in the Older Adult group. 
 
4.5 Descriptive and Inferential Statistics of Clinical features and BCR-ABL1 gene  
 Table 6 show the frequencies of clinical features and test of association with the BCR-ABL1 
gene. Cases which showed the presence of a clinical features are indicated by ‘Yes’ and ‘No’ if 
otherwise. 
 
 
 
 
 
 
 
 
 
 
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Table 6: BCR-ABL1 FUSION GENE AND CLINICAL FEATURES 
               P-value 
Clinical Feature                 BCR-ABL1 Total (Fisher 
RESULTS exact test) 
Negative Positive 
 No 6 4 10  
LYMPHADENOPATHY 60% 40% 100.0%  
 Yes 6 1 7 0.338 
85.7% 14.3% 100.0% 
 No 9 4 13  
SPLENOMEGALY 69.2% 30.8% 100.0%  
Yes 3 1 4  
75.0% 25.0% 100.0% 0.67 
 No 8 4 12  
HEPATOMEGALY 66.7% 33.3% 100.0%  
Yes 4 1 5 0.528 
80.0% 20.0% 100.0%  
 
 Total 12 5 17  
70.6% 29.4% 100.0% 
 
From the Chi-Square analysis above, there is a likelihood of no significant association between 
BCR-ABL1 gene positivity and lymphadenopathy, splenomegaly and hepatomegaly of study 
cases. (P-values = > 0.05). 
 
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4.6 Descriptive and Inferential Statistics of Haematological parameters 
Table 7: BCR-ABL1 FUSION GENE AND HAEMATOLOGICAL PARAMETERS 
 
P-value 
 BCR-ABL1 MW-U 
RESULTS N Mean Std. Deviation test 
WBC Count Negative 12 73.53×109/L  138.87×109/L     0.879  
Positive 5 36.83×109/L 39.21×109/L  
Hb Concentration Negative 12 6.63g/dL 1.82g/dL    0.506 
Positive 5 7.26g/dL 1.34g/dL  
BLAST Negative 11 82.18% 28.16   0.851 
PERCENTAGE 
Positive 4 73.00% 29.72  
PLATELET Count Negative 12 74.33×109/L 59.86×109/L   0.721 
Positive 5 54.60×109/L 37.75×109/L  
 
From the table above, From the table above, the mean haemoglobin concentration for BCR-ABL1 
positive cases was higher than that for the negative cases, whereas the mean white blood cell 
count, bone marrow blast percentages and platelet counts were lower in BCR-ABL1 positive 
cases than in the negative cases. However, the Mann-Whitney test p-values are >0.05, hence no 
significant association exist between BCR-ABL1 gene positivity and these parameters. 
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4.7 Descriptive and Inferential Statistics of BCR-ABL1 gene and Clinical Outcome 
CLINICAL OUTCOME 
Table 8: BCR-ABL1 FUSION GENE AND CLINICAL OUTCOME 
FISH RESULTS 
 Negative Positive Total 
CLINICAL Mortality Frequency 3 1 4 
OUTCOME 
% within Mortality 75.0% 25.0% 100.0% 
Default  Frequency 2 1 3 
% within Default 66.7% 33.3% 100.0% 
Undetermine   Frequency 7 3 10 
d 
% within Undetermined 70.0% 30.0% 100.0% 
Total Frequency 12 5 17 
% within CLINICAL 
70.6% 29.4% 100.0% 
OUTCOME 
 
 
On the basis of inability to determine the clinical outcome 13 out of 17 study samples 
(undertermined =10, default = 3), no inferential assumption will be draw for clinical outcome. 
 
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4.8 Features of the BCR-ABL1 Positive Cases  
The table below features of the BCR-ABL1 positive cases in this study 
Table 9:  FEATURES OF BCR-ABL1 POSITIVE CASES 
 CASE 1 CASE 2 CASE 3 CASE 4 CASE 5               
BCR-ABL1 results Positive Positive Positive Positive Positive 
Age 16 24 36 16 15 
Sex Female Male Male Male Male 
Blast percentage 36 95 - 66 99 
WBC count 1.60 88.4 67.04 24.76 2.33 
Platelet count 12 109 27 67 58 
Haemoglobin 5.8 8.5 8.5 7.6 5.9 
concentration 
Lymphadenopathy No No No No Yes 
Splenomegaly No No No Yes No 
Hepatomegaly No No No Yes No 
Treatment Undetermined Undetermined Defaulted Undetermined Death 
outcome 
Fusion pattern Double Double double double single 
Percentage of 37 8 46 9 20 
fusion signals 
                                                                
 
 
 
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   CHAPTER 5 
 DISCUSSION AND CONCLUSION 
The results of this study show that the BCR-ABL1 gene fusion is present in nearly one-third of 
adult cases of ALL diagnosed in the Haematology Department of the Korle Bu Teaching 
Hospital which were tested. Additionally, the findings suggested no statistically significant 
association between positivity for this chimeric fusion gene and clinical features, haematological 
parameters.   
  
5.1 Frequency 
The findings of this study show that, the BCR-ABL1 fusion gene is present in nearly one third 
(29.4%) of adults diagnosed with acute lymphoblastic leukaemia at the Korle Bu Teaching 
Hospital which were tested.   
 
The frequency of 29.4% in this study is consistent with the prevalence rates 28.3%   in the study 
conducted by Chopra et al. in India and the 11-29% Mrozek et al. in a review of adult ALL 
studies in USA, UK and France. However, it is higher than the 12.5% value obtained by Ajuba et 
al. in Nigeria.  The wide difference in frequency between this study and that of Ajuba et. al (both 
conducted in Sub-Saharan Africa) could arise from the fact that the cases considered in this study 
were adults whereas the subjects for Ajuba et al.  were both children and adults. The frequency 
of the BCR-ABL gene in ALL is age dependent being higher in adults than children as shown by 
Mrozek et al.   The frequency in this study is in contrast to the study in Saudi Arabia  in which 
none of the 16 adult ALL cases was positive for the BCR-ABL fusion gene El-Sissy ET (El-
Sissy et al., 2006). Ethnic difference may possibly account for the disparities as Ariffin et al 
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showed variation in frequency between ethnic groups in their study in Singapore involving 
Indians, Malays and Chinese.  
5.2 Age and Gender 
There was no significant association between BCR-ABL1 positivity and the age groups of cases 
tested in this study (AYA and older adult). However, all the BCR-ABL1 positive cases belonged 
to the adolescent and young adult (AYA) group. This contrast the report of increased frequency 
of BCR-ABL1 with increasing age reaching 42% to 44% beyond age 44 in Germany (Thomas 
Burmeister et al., 2008) . The exact reasons for the occurrence of the fusion gene in only the 
AYA group is not clear. However, the decreased representation of the older adult group in this 
study cannot be ruled out.  Ghana has a young population with 58.5% aged 0 to 24 years and 
only 11% beyond age 50 years. This contrasts the population structure of advanced countries 
such as  U.S.A, Germany and Japan where the population is relatively old with respective 
proportions of  33.0%, 24.7% and 22.5% in the 0 to 24 years age group  whereas 34.2%, 40.3% 
and 45% are beyond age 50 years(Nations, 2017). The lower proportions old age population in  
Ghana which possibly reflected in this study can be attributed to the fact that there is  relatively 
low life expectancy at birth (61.3 years) in Ghana compared to that of the U.S.A, Germany and 
Japan which is 78.9, 80.8 and 83.6 years respectively (United-Nations., 2017).  
 
Similarly, this study suggests no significant association between BCR-ABL1 positivity and sex 
although 4 out of 5 cases of the BCR-ABL1 positive cases were males. This may be explained by 
the low proportion of females (23.5%) in this study. 
These findings of no significant association between BCR-ABL1 positivity and age as well as 
gender in study cases confirms the study of Hamid and Bokharaei in Iran. 
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5.3 Clinical Features 
Secondly, the findings of this study suggest no significant association between BCR-ABL1 
positivity and clinical features (lymphadenopathy, splenomegaly and, hepatomegaly) of study 
cases.    
Similarly, Westbrook et al. in a Cancer and Leukaemia Group B study in USA found no 
statistically significant association between BCR-ABL1 positivity and these clinical features.  
Studies involving clinical features and BCR-ABL1 positive ALL from Africa (especially West 
Africa) are virtually not available. 
  
Of the clinical features considered in this study, Lymphadenopathy was present 41.2% of all the 
cases in this study of which 14.3% were BCR-ABL1 positive whereas 23.5% of the cases studied 
showed splenomegaly of which one quarter was BCR-ABL1 positive. While hepatomegaly 
evident in 35.7% of study cases with exactly one fifth showing BCR-ABL1 positivity, no incident 
of mediastinal mass was reported in this study.  The frequencies of the organomegalies are lower 
than what has been reported in the study by Elbossaty et al.  in Egypt in which 
lymphadenopathy, splenomegaly and hepatomegaly occurred in 58, 58 and 54% of adult ALL 
patients. However, it is consistent with the findings in Netherlands by Daenen et al. in which 
organomegalies (lymphadenopathy, splenomegaly and hepatomegaly) were less prevalent with a 
combined frequency of 40% in adult ALL. 
 
5.4 Haematological Parameters 
The mean white blood cell count, bone marrow blast percentages and platelet counts were lower 
in BCR-ABL1 positive cases than negative (36.83, 73.00 and 54.60 versus 73.53, 82.18 and 74.33 
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respectively). Contrastingly, the mean haemoglobin concentration for BCR-ABL1 positive cases 
was higher than in negative cases (7.26 versus 6.62 respectively). However, no significant 
association was established between these mean values of these haematological parameters and 
BCR-ABL1 positivity (P = 0.879, 0.721, 0.506 and 0.851 for WBC counts, platelets counts, 
haemoglobin concentration and blast percentage respectively). 
The results of this study confirm the findings of Ajuba et al. in Nigeria in which no statistically 
significant association was observed between BCR-ABL1 positivity and white blood cell count, 
platelet count and haemoglobin concentration (P = 0.187, 0.658 and 0.303 respectively). 
 The findings of no statistically significant difference white blood cell count and haemoglobin 
concentration in this study is in contrast to the findings in the study by Gleiβner et al. in 
Germany in which BCR-ABL1 positive cases showed statistically significantly higher WBC 
count and haemoglobin concentration than BCR-ABL1 negative cases. However, this study found   
no statistically significant difference in platelet counts and blast percentage in BCR-ABL1 
positive and negative cases as was reported by Gleiβner et al. 
 
All the cases in this study had severe to moderate anaemia with haemoglobin concentration 
ranging from 3.7 to 8.7g/dL. Also, with the exception of one, all cases in the study 
(approximately 94%) showed thrombocytopenia. These observed frequencies and degree of 
anaemia and thrombocytopenia is higher compared to developed countries based on studies 
conducted in Denmark and Italy   (Chiaretti et al., 2013; Toft, Schmiegelow, Klausen, & 
Birgens, 2012). This may result from delayed presentation of the patients in this study to the 
Haematology clinic and hence late diagnosis and treatment thus allowing leukaemia cells 
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sufficient time to suppress normal erythropoiesis. This challenge is common in developing 
countries such as Ghana due to inadequate number of Haematology centers.   
 
5.5 Clinical Outcome 
  The clinical outcome of most of the cases which were studied could not be determined. This 
arose from the fact that a total of 13 (76.5%) of study cases who constituted the default and 
undetermined categories ceased clinic attendance based on financial constraint or unknown 
reasons respectively. 
 
5.6 Limitations of the Study 
 Inadequate filing system (for slides and folders), unavailability of corresponding clinical and 
laboratory data from folders and laboratory and reduced number of good quality bone marrow 
aspirate slides prevented one from obtaining the calculated sample size of 30; seventeen cases 
were thus studied. 
Secondly, the overall survival of majority study participants could not be determined due to 
truncated clinic attendance. 
 
5.7 Conclusion 
With a frequency of 29.4%, the BCR-ABL1 fusion gene is an important molecular genetic lesion 
in adult ALL cases in this study. There was no significant association between BCR-ABL1 
positivity and clinical features as well as haematological parameters of cases which were studied.   
 
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As no information of the BCR-ABL1 gene fusion in acute lymphoblastic leukaemia is available in 
Ghana due to the fact that no research has been carried out on it, this study thus provides an 
initial information on its presence, and association with clinical features and treatment outcome 
for stakeholders involved in the diagnosis and treatment of acute lymphoblastic leukaemia to 
make an informed decision with regard to the need for a bigger study. 
The BCR-ABL1 fusion gene is thus expressed in adult acute lymphoblastic leukaemia cases seen 
in the country and has no significant association with the clinical features and haematological 
parameters of the disease the cases which were studied. A larger study will be needed to mak a 
determination concerning the modification of treatment regimen for adult BCR-ABL1 positive 
ALL. 
 
5.8 Recommendation 
The investigator of this study suggests a larger multicenter prospective study in Ghana on BCR-
ABL1 positive acute lymphoblastic leukaemia involving the characterization of the associated 
molecular signatures such as the IKZF1 and the PAX genes so as to throw more light the 
expression of the disease in Ghanaian patients. 
 
 
  
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APPENDIX A 
DATA ABSTRACTION FORM (Pages 21-22) 
Personal Information 
 PATIENT UNIQUE ID NUMBER:                                                                 
  AGE:                                      SEX:                                                                   DATE OF 
DIAGNOSIS: 
  
Clinical Feature 
 “A” will be circled for each of the clinical sign numbered 1-4 below if they are present and “B” 
if not indicated. Any others sign must be written in the space provide in front of number 5 below. 
1. Lymphadenopathy   -          A.    YES                              B.     NO 
2. Mediastinal mass      -          A.    YES                              B.     NO   
3. Splenomegaly            -          A.    YES                              B.     NO          
4. Hepatomegaly           -          A.    YES                              B.     NO 
5. Other signs                 -          ………………………………………………………………… 
 
 
 
 
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Clinical Outcome 
Date of diagnosis (A): 
Last date seen at clinic (B): 
Outcome of treatment: A. remission B. death C. default D. failure    
 E. others: ……………………………………… 
Survival duration = duration between A and B= 
 
Haematological Parameters 
PERIPHERAL BLOOD: 
White cell count -   
 Platelet count - 
Haemoglobin concentration – 
 
BONE MARROW: 
Blast percentage: 
 
 
 
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FISH results for BCR-ABL1 fusion gene: 
“A “or “B “will be circled   below if bone marrow aspirate slide is BCR-ABL1 positive or 
negative respectively. 
A. POSITIVE                       B. NEGATIVE 
 
Percentage score (If FISH results above is positive) - ..................% 
 
 
 
 
 
 
 
 
 
 
 
 
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APPENDIX B 
  TABLES OF RESULTS OF WBC COUNTS, PLATELET COUNTS, HAEMOGLOBIN 
CONCENTRATION AND BLAST PERCENTAGES 
Table 1: WBC RESULTS 
    WBC Valid Cumulative 
(×109/L) Frequency Percent Percent Percent 
Valid .95 1 5.9 5.9 5.9 
1.60 1 5.9 5.9 11.8 
1.61 1 5.9 5.9 17.6 
2.33 1 5.9 5.9 23.5 
6.41 2 11.8 11.8 35.3 
7.20 1 5.9 5.9 41.2 
9.60 1 5.9 5.9 47.1 
16.43 1 5.9 5.9 52.9 
24.76 1 5.9 5.9 58.8 
38.58 1 5.9 5.9 64.7 
42.50 1 5.9 5.9 70.6 
67.04 1 5.9 5.9 76.5 
88.40 1 5.9 5.9 82.4 
106.00 1 5.9 5.9 88.2 
160.70 1 5.9 5.9 94.1 
485.91 1 5.9 5.9 100.0 
Total 17 100.0 100.0  
 
 
 
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TABLE 2: PLATELET COUNTS 
Valid Cumulative 
 PLT (×109/L) Frequency Percent Percent Percent 
Valid 1.0 1 5.9 5.9 5.9 
8.0 1 5.9 5.9 11.8 
9.0 1 5.9 5.9 17.6 
12.0 1 5.9 5.9 23.5 
27.0 1 5.9 5.9 29.4 
29.0 1 5.9 5.9 35.3 
49.0 1 5.9 5.9 41.2 
58.0 1 5.9 5.9 47.1 
64.0 1 5.9 5.9 52.9 
65.0 1 5.9 5.9 58.8 
67.0 1 5.9 5.9 64.7 
96.0 1 5.9 5.9 70.6 
109.0 1 5.9 5.9 76.5 
122.0 1 5.9 5.9 82.4 
125.0 1 5.9 5.9 88.2 
132.0 1 5.9 5.9 94.1 
192.0 1 5.9 5.9 100.0 
Total 17 100.0 100.0  
 
 
 
 
 
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Table 3: HAEMOGLOBIN CONCENTRATIONS 
Valid Cumulative 
   Hb(g/dL) Frequency Percent Percent Percent 
Valid 3.7 1 5.9 5.9 5.9 
3.8 1 5.9 5.9 11.8 
4.5 1 5.9 5.9 17.6 
5.7 1 5.9 5.9 23.5 
5.8 1 5.9 5.9 29.4 
5.9 1 5.9 5.9 35.3 
6.0 1 5.9 5.9 41.2 
7.2 1 5.9 5.9 47.1 
7.6 2 11.8 11.8 58.8 
7.9 1 5.9 5.9 64.7 
8.0 2 11.8 11.8 76.5 
8.4 1 5.9 5.9 82.4 
8.5 2 11.8 11.8 94.1 
8.7 1 5.9 5.9 100.0 
Total 17 100.0 100.0  
 
 
 
 
 
 
 
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     TABLE 4: BLAST PERCENTAGE 
Valid Cumulative 
 Frequency Percent Percent Percent 
Valid 1.0 1 5.9 6.7 6.7 
36.0 1 5.9 6.7 13.3 
62.0 1 5.9 6.7 20.0 
75.0 1 5.9 6.7 26.7 
79.0 1 5.9 6.7 33.3 
80.0 1 5.9 6.7 40.0 
93.0 1 5.9 6.7 46.7 
94.0 1 5.9 6.7 53.3 
95.0 3 17.6 20.0 73.3 
97.0 2 11.8 13.3 86.7 
98.0 1 5.9 6.7 93.3 
99.0 1 5.9 6.7 100.0 
Total 15 88.2 100.0  
Missing System 2 11.8   
Total 17 100.0   
 
N.B. The blast percentage for two participants were not available. 
 
 
 
 
 
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APPENDIX C 
The reagents for FISH procedure are listed below: 
 
1. Vysis LSI BCR/ABL, dual colour, dual fusion translocation probe set which comprised: 
a. Vysis LSI BCR/ABL, dual colour, dual fusion translocation probe  
b. Vysis LSI /WCP hybridisation buffer 
2. DAPI counterstain 
3. Methanol 
4. Ethanol 
5. 2× Saline – Sodium Citrate (SSC) for molecular biology 
6. 1×Phosphate buffered saline 
7. 1% Formaldehyde  
8. IgePal (Octyl phenyl-polyethylene glycol) for molecular biology 
9. Rubber cement (ELMER’S) 
10. 0.01N HCL 
11. 0.2N HCL 
12. Pepsin 
13. Double distilled water (ddH2O) 
 
 
 
 
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APPENDIX D 
 
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