CONCEPTUAL FRAMEWORKS
7.2 STAGING THE PERFORMANCE
GENERAL AIM
The general aim of the study was to determine the pattern of cerebral blood flow velocities using Transcranial Doppler ultrasonography in sickle cell anaemia patients aged 2-16 years in steady state attending the Paediatric Sickle Cell clinic in Lagos University Teaching Hospital (LUTH), Lagos.
SPECIFIC OBJECTIVES The specific objectives were to:
1. Determine the pattern of cerebral flow velocities in sickle cell anaemia patients aged 2-16 years and the proportion of those with high cerebral blood flow velocities (CBFV).
2. Compare the cerebral blood flow velocities of sickle cell anaemia patients with controls aged 2-16 years.
3. Determine the relationship between socio-demographic variables [age, social economic class and gender], clinical parameters [systolic hypertension, history of acute chest crisis], haematological variables [white cell count, platelet, and haemoglobin], oxygen saturation and cerebral blood flow velocities in sickle cell anaemia and HbAA aged 2-16 years
34 SUBJECTS AND METHODS
STUDY LOCATION
The study was study conducted at two locations in Lagos. Lagos state is located in the western part of Nigeria and has 20 Local Government Areas with an estimated population of 9 million..128 _ENREF_158The specific locations were:
1] The Paediatric Haematology Clinic of LUTH Idi-Araba Lagos. LUTH is a 760 bed tertiary centre providing health care to Lagos and its environs. The clinic runs every Thursday providing care to children up to eighteen years with sickle cell disease and other haematological diseases. It is run by three consultants and at least five residents. The researcher and all the trained research assistants were at the weekly clinics to give health talks on TCD and obtained all the information for the study pro-foma [bio data, family and social histories and anthropometric examination] after consent and assent were obtained from both parents and participants respectively. The clinic has a weekly average of 10-12 new patients with routine attendance by 45-52 patients weekly. As at September 2015, the clinic had approximately 2000-2500 registered patients. The TCD examination is usually done at Sickle Cell Foundation-Nigeria, Idi-Araba, Lagos which is directly opposite Lagos University Teaching Hospital (LUTH), Idi-Araba.
2] The Sickle Cell Foundation-Nigeria, Idi-Araba, Lagos. This is a non- Governmental organization (NGO) just opposite LUTH. The Foundation provides services such as genetic counseling to persons with sickle cell disease and their families. The foundation has four sonologists who perform the transcranial Doppler scan daily for all patients with sickle cell anaemia aged two years and above. The clinic at the foundation runs daily except on weekend with monthly attendance of 200 children with sickle cell disease.
35 STUDY POPULATION
The subjects for the study were 315 children with sickle cell anaemia patients aged 2-16 years and the 315 controls aged 2-16 years who were apparently healthy age and sex matched children with genotype AA who attended the Well Baby and Out-Patients Clinic in LUTH.
INCLUSION CRITERIA FOR SUBJECTS
The following inclusion criteria were used to select children with SCA into the study:
Sickle cell anaemia patients in steady state [a baseline state in which SCA patients are relatively asymptomatic for at least four weeks before recruitment]129 aged two to sixteen years
Informed consent obtained from parents and assent from children older than seven years
INCLUSION CRITERIA FOR CONTROLS
The following inclusion criteria were used to select children with HbAA into the study:
Apparently healthy children from Well-Baby Clinics and Out-patients Clinics (children attending the under-5 clinic for growth monitoring and immunization and older children with minor ailments) with haemoglobin genotype AA aged two to sixteen years
Apparently healthy children well Baby Clinics and Out-Patients Clinics with no evidence of recent infection or acute illness within four weeks prior to the study (children attending the under-5 clinic for growth monitoring and immunization and older children with minor ailments).
Informed consent were obtained from parents and assent from children older than seven years EXCLUSION CRITERIA FOR SUBJECTS AND CONTROLS
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The following exclusion criteria were used to exclude children with HbSS and HbAA into the study:
History or evidence of neurological disease [previous stroke, meningitis].
Subjects on hydroxyurea therapy or chronic blood transfusion.
Recent blood transfusions [within 30 days for subjects and 120 days for controls prior to study] and acute illness [such as painful crisis, fever, infectious disease].
Hospitalization within four weeks prior to screening.
Refusal to give consent/ assent.
Subjects and controls with incomplete data.
Children with haemoglobin genotype other than Hb SS or Hb AA
ETHICAL APPROVAL AND CLEARANCE
Ethical clearance and approval of the Health Research and Ethics Committee of Lagos University Teaching Hospital (LUTH), was obtained (See Appendix I) and a revalidation of the ethical approval was sort from the Health Research and Ethics Committee after the expiration of the first approval. (See Appendix I) Also permission was obtained from the Chairman, Sickle Cell Foundation-Nigeria prior to commencement of the study. (See Appendix II) The objectives and details of the study were explained to all parents and patients before their written consents and assent were obtained. The subjects recruited benefitted from the study having their transcranial Doppler scan done and haematological parameters assayed free of charge while the controls had free haemoglobin electrophoresis and free medical checkup, free consultation and counseling
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done. Also those with abnormal results were evaluated by the researcher and when necessary, were referred for further appropriate care.
STUDY DESIGN
The study was descriptive cross sectional
SAMPLING METHOD
Systematic sampling method was used to recruit the children with sickle cell anaemia and controls. The total number of patients registered as of September 2015 was an average 2000 children with monthly attendance of 45-52 children. Thus the sample interval was total number of patient/ number of monthly attendance by patients which means 315/52 which was equal to 6 as the sampling interval. Prior to the systematic sampling, simple random sampling was done by balloting between number 1 and 6. The clinic register was used for the sampling.
Consequently, the starting point for sampling was number 3 following the balloting and the subsequently the patients were recruited as follows numbers; 3 (1st subject), 9, 15, 21………..1890th (315th subject) Also same was done for the controls.
However recruitment were from Well Baby Clinic with an average of 40 to 60 children seen monthly and out -patient clinic with an average of 30 to 40 children monthly. Total of 180 children with HbAA genotype were recruited from Well Child Clinic and 135 children from Out-Patients Clinic with sample interval of 3 for both studied populations. The clinic register (a book where patients are sequentially registered on their presentation to clinic with a serial number )was also used and a simple random sampling was done between 1 and 3 and the starting
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nth was 1 and subsequently 1st---4th---7th---10th---480th (180thcontrol) from Well Child Clinic and -1st---4th---7th---10th ---405th (135thcontrol) from Outpatient Clinic.
SAMPLE SIZE DETERMINATION
Using the standard statistical method for finite population, the minimum sample size was calculated as below130:
N= Z2Pq d2
Where N= Desired sample size
Z= Normal standard deviation for required level of confidence, and this is set at 1.96 which corresponds to the 95% confidence interval.
P= Prevalence of the condition under study q= 1-P
d= Degree of accuracy, which be set at 0.05 for this study.
The prevalence of abnormal velocity in the study of Lagunju et al11 will form the basis of my calculations (i.e. P= 0.244).
Using the formula above, the minimum sample size needed is calculated as follows:
N= (1.96)2(0.244) (1-0.244) (0.05)2
= 283
The minimum sample size needed were 283 for subjects and 283 for controls.
However, with the addition of a projected attrition rate of 10%, the sample size became 315 in each group.
39 Recruitment of Subjects and controls
The study was carried out at the Paediatric Haematology, Well Child and Outpatient Clinics of LUTH, Idi-Araba, Lagos between September 2015 to May 2016. All cases of sickle cell anaemia aged 2 years to 16 years whose genotype have already been confirmed by haemoglobin electrophoresis were seen at the Paediatric Haematology Clinic of LUTH over a period of nine months. The control group who were age and sex matched patients with Hb AA genotype confirmed by haemoglobin electrophoresis were recruited from Well-Baby and Out-Patients Clinics in LUTH. Some trained research assistants helped in the study. These research assistants were trained by the researcher for 10 days on recruitment of patients, questionnaire collection, measurement of anthropometry, blood sample collection and data collection.
At every clinic session, all medical chats of each child were reviewed to ascertain eligibility by the researcher and research assistants. Informed consent (See Appendix III A and B) and assent (See Appendix III C) were obtained from the parents or guardians of eligible children as needed after they have been counseled by the researcher on research objectives. Those who meet the inclusion criteria were recruited for the study. Systematic samplings were used in recruitment of both subjects and controls. A profoma (See Appendix IV) designed for the study was used to record the information obtained. Then semi-structured questionnaires were administered by the research assistant. The following information was obtained from both subjects and controls that satisfied the inclusion criteria and recorded in the study profoma (See Appendix IV): serial number, age, gender, parents’ address, tribe, religion, age, marital status, occupation and education of parents, haemoglobin genotype and those that did not were excluded. Review of past medical histories was carried out to identify the complications of sickle cell anaemia that the child has suffered previously by the researcher: frequency of painful crises in the last one year prior to study and
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previous hospitalizations for painful crisis, and previous blood transfusions in life within one year but more than a month prior to the study, family history of stroke, medications the child is on, number of siblings with HbSS, past history suggestive of acute chest crisis( those who had cough, fever, chest pain, bone pain or admitted for crisis) and other clinical problems the child has.
Demographic data and haemoglobin genotype was also obtained and recorded for all the children that satisfied the inclusion criteria for the control group. Socio-economic stratification of the family of each child was determined using Oyedeji criteria and method (See Appendix V) which utilizes both parents education and occupation.131
Height was measured by the research assistant in centimeters (cm) using a seca Leicester floor mounted stadiometer. For height measurement, the child stood barefooted, erect with the occiput, shoulder blades, buttocks, calf and the heel making contact with the stadiometer and the head in the Frankfurt plane. Accuracy of the secaTM scale was checked by recalibrating each study day with known five kilogram weight.
Clinical examination was carried out on each of the subject and control in the clinic after recruitment by the researcher. All the children had arterial oxygen saturation (SaO2) measured using a hand held pulse oximeter (model AH-M1 by A care technology) by research assistant. The blood pressure was taken after patient had sat down for 5 minutes and the right arm supported on a horizontal surface at the level of the heart by research assistant. The blood pressure reading was taken using mercury sphygmomanometer and was measured once.While in sitting position, a cuff with an inflatable bladder width of at least 40% of the child’s mid upper arm circumference that covered 80% of the upper arm length was applied around the right arm. Measurements were taken once in sitting position. Reading was recorded to the nearest 2mmhg, the first Korotkoff (K1) was the systolic while the fifth Korotkoff sound (K5) was taken as diastolic pressure.132 The blood
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pressure of each subject and control was compared with that of general population as published by the National High Blood Pressure Education Program.132
Venepuncture was carried out by the research assistants and during the sample collection; the site was cleaned with methylated spirit/ povidone iodine. Universal precaution for prevention of contacts with all human blood and bodily fluids was adhered to.133 Approximately three millimeters venous blood was collected from the ante-cubital fossa after tourniquet was applied and was put into the Ethylene Diamine Tetra Acetic (EDTA) container. The vaccum containers were labeled and placed in a cool box containing ice-packs. The samples were protected from light at all times using sheets of black plastic. Samples were transported to department of Paediatrics Haematology laboratory for full blood count estimation on same day (using automated haematology analyzer Mindray, BC-2800). The fresh samples in EDTA containing bottles were used for determination of packed red cell (PCV), haemoglobin (Hb), white cell count (WBC), and platelet prior to the TCD ultrasound scan by laboratory scientist. Haemoglobin electrophoresis results were retrieved from the case notes to confirm the haemoglobin genotype for subjects and those whose haemoglobin genotype results were not available had it repeated from the blood samples drawn for full blood count estimation. For the controls, those whose haemoglobin electrophoresis was not HbAA were discarded and replaced by the selection of the next control.
The haemoglobin genotype of some of the subjects and all of the controls were confirmed using cellulose acetate haemoglobin electrophoresis at pH 8.6 as described by Dacie and Lewis.134 About 50 ul of EDTA blood samples of subjects and controls were lysed with equal volume of water and haemogobin separated out with carbon tetrachloride. Lysed samples of patients and controls and known AA, AS, SC, and SS controls were placed on cellulose acetate paper in batches using applicator. The paper was placed in the electrophoretic tank containing tris-buffer, pH 8.6, electric
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current was applied. All samples and AA, AS, SC, and SS controls samples migrated from negative to positive pole at 400volt. Haemoglobin separation occurs within 4-6 minutes, haemoglobin bands were compared with known controls analysed by laboratory scientist and few by the researcher.
The expenses were bore by the researcher.
Determination of Socio-economic class
For the purpose of the study, social classification was done using the scheme proposed by Oyedeji.131(See Appendix V). The social class was stratified into five classes based on occupation and maximal educational attainments of both parents (and their substitute) as described by Oyedeji.131 The mean of the fours scores, ( two for father and two for mother) to the nearest whole number, was assigned to the child.131 Scores of I-111 were awarded as upper socioeconomic class and IV and V were awarded as lower socioeconomic class.131
Determination of Blood Pressure Using World Health Organization (W.H.O) Blood Pressure Tables132
Blood pressure standard based on gender, age, and height provide a precise classification of blood pressure according to body size. 132 The height percentile was determined using CDC growth chart (See Appendices VI A and B) Each child’s blood pressure was compared with the numbers provided in the tables for boys and girls (See Appendices VII A and B respectively) according to the child’s age and height percentile.
Blood Pressure Classification
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The blood pressure was classified into four groups and includes the following:
Normal Blood Pressure: if the blood pressure is <90th ≥ 50th percentile Low Blood Pressure: Blood Pressure <50th percentile
Pre-Hypertension: Blood Pressure ≥90th but ≤ 95th percentile Hypertension: Blood pressure ≥95th percentile
TRANSCRANIAL DOPPLER [TCD] EXAMINATION
The STOP protocol was used for the TCD examinations. The researcher received four weeks training on TCD techniques under Dr [Mrs.] Soyebi, a consultant radiologist at LUTH and the sonologists at the Sickle cell Foundation prior to the commencement of the study. . A pre-test was done prior to commencement of study. A pilot study was done to minimize errors and to determine the efficacy of the knowledge gained by the researcher during the training. This involved 7 subjects with HBSS and 11 controls who were scanned independently by the investigator first and then by the sonologist. The velocities were compared after the scans were completed. A concordance of 85% was achieved before the main study commenced.
All scans were done at the Sickle cell, Foundation centre, Lagos using a Doppler-Box Tm with DWL Doppler system and QL software. The technique was similar to that for the STOP trial.16 The subjects and controls were kept calm but awake to avoid the resultant increased carbon dioxide of sleep that could lead to increased cerebral blood flow velocity. Cerebral blood flow velocities in the major cerebral arteries were measured through trans-temporal windows with a 2MHZ hand–
held probe or transducer attached to the Doppler Machine (DB-0563, Compumedics, Germany) using the non- imaging TCD techniques. The examinations were carried out in a quiet room
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dedicated for only TCD at the Sickle cell foundation Centre. The procedure was explained to the patient and caregiver, including the need for the child to remain awake and cooperative during the examination. The younger children (2-3 year olds) were also patiently explained to and the procedure demonstrated. Their caregivers were involved in reassuring them to ensure their cooperation as they were allowed in the TCD room.
With the patients lying supine and the examiner seated at the head of the couch with his arms supported and the instruments within easy reach. Sitting at the head of the couch enabled the examiner easy access to the two temporal regions. Thus with the patient in a supine position and the head stabilized, a 2MHz transducer coated with ultrasound gel was held against the temporal part of the head and with appropriately maneuvered probe angulations and position, the depth of the instrument and the ultrasound volume to measure the Time-Averaged Mean of the Maximal Velocity (TAMMV) in centimeters per second [cm/sec], scanning in 2mm increments along (the major arterial segments of the Circle of Willis) Middle Cerebral Artery (MCA) or distal Internal Carotid Artery (ICA) and the Anterior Cerebral Artery (ACA). Measurements were taken from the optimized Doppler spectral waveforms during a minimum of three cardiac cycles. Multiple measurements were taken at varying depths on either side and for each vessel between 40mm and 60mm. Added to the explanation, the 2 MHz hand held transducer, coated with ultrasound gel was applied to the right trans-temporal window located anterior to the tragus and superior to the zygoma. The middle cerebral artery (MCA) with a forward flow on the spectral display was identified and tracked at 2mm depth intervals, with the signal optimized at each depth and the velocities (TAMMV) recorded at each depth. After completion of the study on the right side, the same procedure was carried out on the left side on all the subjects. The subjects in the control group also had TCD scans as described above. Velocity was recorded as a spectrum with time on
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the x-axis and velocity in centimeter per second on the y-axis. The highest recorded velocity for each artery was assumed to be the most representative and thus was recorded as Time-Average Mean of the Maximum Velocity [TAMMV]. All the measurements were recorded.
The grading of risk classification was in accordance with the protocol for STOP trial.16 This was based on the highest TAMMV in the ICA or MCA or ACA on either cranial side. Thus, TAMMV values< 170cm/sec, 170-199cm/sec and > 200cm/sec were regarded as indicating: normal cerebral flow velocity (standard stroke risk), conditional risk, and abnormal cerebral blood flow velocity (high stroke risk), respectively.
Subjects with elevated velocities of greater than 170cm/sec had a repeat TCD scan and if still abnormal, were referred to Paediatric Haematology Clinic of LUTH for further management after the results had been explained to them. For every 10 results reported by the researcher, the sonologist re-scanned at least one for validation of results.
DATA ANALYSIS
Data generated were validated by researcher using cross reference and second look for accuracy and consistency to avoid mistakes on spread sheets and analyzed using appropriate Statistical Package for Social Sciences [SPSS] version 20. Data cleaning was done by running a frequency distribution to detect incorrect entries which were then corrected. The baseline socio-demographic and clinical characteristics of participants were compared between the HbAA and HbSS groups.
The mean ± standard deviation and ranges was used to report continuous variables. Frequencies and percentages were computed for the categorical variables. Means of continuous variables were compared using the student T test and ANOVA while associations between categorical variables
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were done using Chi square. Relationship between the demographical, clinical, haematological variables and cerebral blood flow velocities (TAMMV) were determined using linear regression analysis and correlation analysis. The level of statistical significance was set at P-value of less than 0.05. Results were presented in tables, charts and graphs. Multivariate linear regression analysis was utilized in determining factors predictive of abnormal cerebral blood flow velocities. The level of statistical significance was set at P-value of less than 0.05 for all the analyses.
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