|Year : 2015 | Volume
| Issue : 1 | Page : 3-10
Clinical, hematological, and cytogenetic profile of aplastic anemia
Subhajit Das MD 1, Vijai Tilak1, Vineeta Gupta2, Anup Singh3, Mohan Kumar1, Amit Rai4
1 Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Paediatrics, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Department of General Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
4 Department of Centre for Genetic Disorders, Faculty of Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
|Date of Submission||07-May-2014|
|Date of Acceptance||21-Nov-2014|
|Date of Web Publication||24-Apr-2015|
63 F/1 B/1 Tollygunge Road, Kolkata 700033, West Bengal
Source of Support: None, Conflict of Interest: None
Background Aplastic anemia manifests as peripheral blood cytopenias due to inability of the hematopoietic bone marrow to produce blood cells.
Aims and objective The aim of this study was to investigate the differences in the clinicohematological profile of pediatric and adult aplastic anemia patients, if any, to establish the necessity of bone marrow biopsy for its diagnosis and to detect any chromosomal abnormality in the patients.
Setting and design This study was carried out in the Departments of Pathology, Paediatrics, and General Medicine, and Centre for Genetic Disorders, in a university teaching hospital over 24 months.
Materials and methods A total of 42 patients were diagnosed combining the clinical features, peripheral blood counts, and bone marrow examination. Of them, 24 patients were evaluated for cytogenetic abnormality.
Statistical analysis All the observations were evaluated using simple and basic statistical tools - for example, range, mean, and median.
Results Occurrence of severe anemia and frequency of leukopenia and absolute neutropenia were more common in children. The degree of leukopenia and absolute neutropenia was more severe in children. Because of frequent occurrence of blood tap and dry tap, diagnosis in many cases depended solely on bone marrow biopsy findings. All the core biopsy specimens floated after proper decalcification. Lymphocyte culture failed in nine cases, and the remaining 15 cases showed no cytogenetic abnormality.
Conclusion Bone marrow biopsy is mandatory for diagnosing aplastic anemia. The findings of core biopsy floatation and its correlation with marrow cellularity and absence of chromosomal abnormality in aplastic anemia need larger studies to give any statistically significant opinion.
Keywords: aplastic anemia, bone marrow biopsy, chromosomal abnormality, peripheral blood cytopenia
|How to cite this article:|
Das S, Tilak V, Gupta V, Singh A, Kumar M, Rai A. Clinical, hematological, and cytogenetic profile of aplastic anemia. Egypt J Haematol 2015;40:3-10
|How to cite this URL:|
Das S, Tilak V, Gupta V, Singh A, Kumar M, Rai A. Clinical, hematological, and cytogenetic profile of aplastic anemia. Egypt J Haematol [serial online] 2015 [cited 2020 Jan 21];40:3-10. Available from: http://www.ehj.eg.net/text.asp?2015/40/1/3/155779
| Introduction|| |
Aplastic anemia, an unusual hematologic disease, falls in the spectrum of human bone marrow failure syndromes. It is a disorder in which there is a partial or complete shutdown of normal hematopoiesis in the bone marrow. In patients with aplastic anemia, the bone marrow lacks hematopoietic precursor cells and is partially or completely replaced by fat cells. The paucity of normal hematopoiesis is reflected by cytopenias affecting all the three lineages.
The origin of aplastic anemia is considered to be heterogenous. Several studies have been conducted so far to identify genetic, environmental, and other factors that may lead to marrow failure, and on the basis of these studies aplastic anemia is broadly classified into inherited and acquired aplastic anemias  .
The incidence of aplastic anemia has been progressively increasing over the last few decades because of increased exposure to drugs, having potential to cause depression of bone marrow, and to radioactive substances. At present, the overall incidence of the disease in general population is three to six per million, considering both inherited as well as acquired aplastic anemia  . The age distribution is bimodal, with peaks in adolescents and elderly  .
There is a paucity of data on aplastic anemia from India because of limited number of studies. Our study was conducted to describe the clinicohematological features and cytogenetic profile of 42 aplastic anemia cases diagnosed at our center during the study period.
| Materials and methods|| |
This prospective study was carried out in the Departments of Pathology, Paediatrics, and General Medicine, Institute of Medical Sciences, Banaras Hindu University, and Centre for Genetic Disorders, Faculty of Science, Banaras Hindu University over a period of 24 months (2011-2013).
A total of 42 patients ranging in age from 3 to 86 years, with a male to female ratio of 2.2: 1, satisfying the inclusion criteria for aplastic anemia were included in this study. The inclusion criteria were as follows  :
- Peripheral blood showing at least two out of three of the following:
- Hemoglobin less than 10 g/dl or hematocrit less than 30%.
- Total leukocyte count less than 3.5 × 10 9 /l or granulocyte count less than 1.5 × 10 9 /l.
- Platelet count less than 50 × 10 9 /l.
- Bone marrow biopsy showing the following:
- Decrease in cellularity with absence or depletion of all hematopoietic cells less than 25%.
- Absence of significant fibrosis or neoplastic infiltration.
Patients having a history of receiving chemotherapy or radiotherapy for any illness over the past 12 months were excluded from the study. Peripheral blood samples from five age-matched unrelated controls were collected for cytogenetic work up.
Informed consent was obtained from patients, and from guardians in the case of minors, and also from controls. The study was approved by the ethical committee of the hospital.
The following laboratory investigations were carried out in all patients:
- Complete blood count, including reticulocyte count;
- Peripheral blood film examination;
- Bone marrow aspiration, touch imprint, and biopsy;
- Liver function test;
- Serologic testing for HIV, Ebstein Barr virus (EBV), and hepatitis A, B and C;
- Ham test to detect peroxysmal nocturnal hemoglobinuria;
- Basic cytogenetic karyotyping; and
- Chromosomal breakage analysis with mitomycin C(MMC) to diagnose Fanconi anemia.
Collection of bone marrow samples was carried out in the inpatient procedure rooms at the respective departments, under proper aseptic conditions and after administration of a local anesthetic over bilateral posterior superior iliac spines. Pediatric patients were presedated with intravenous ketamine. About 0.5 ml of bone marrow aspirate sample was drawn out in each case and smeared. Trephine biopsy samples were obtained from areas close to the bilateral posterior superior iliac spine. Attempts were made to yield a biopsy length of at least 2.5 cm. Touch imprint smears were prepared by gently touching the core biopsy samples with clean glass slides. The core biopsy samples had undergone fixation in 10% formalin for 24 h and decalcification in 5% formic acid with formalin for 12 h. The floatation status of the core biopsy samples was noted at the end of decalcification. Thereafter, paraffin blocks were made and slides were prepared. All peripheral blood films and bone marrow aspiration and imprint smears were air-dried and stained with Leishman stain. The biopsy slides were stained with hematoxylin and eosin and with reticulin.
A sterile 5 ml syringe was rinsed with heparin, and 3 ml peripheral blood sample was collected and immediately transported (within an hour) to the cytogenetic laboratory.
A volume of 0.5 ml of blood sample was added to 4.5 ml growth media containing RPMI with preadded gentamycin, bovine serum, and phytohemagglutinin and mixed thoroughly. Two such cultures were prepared. MMC was added at a dose of 50 mmol/l to one culture, and both the cultures were incubated for 70 h at 37°C. Thereafter, 6 μl of colchicine was added to both the cultures and incubated again at 37°C for 2 h, and harvesting and slide preparation were performed according to standard methods for preparation of chromosomes  . Slides were stained with Giemsa solution and chromosomal aberrations were recorded. The MMC-induced chromosomal breakages were compared with healthy controls.
| Results|| |
A total of 22 pediatric (<18 years) and 20 adult (≥18 years) patients satisfying the inclusion criteria were included in the study. Among them, 29 were male and 13 were female patients, with a ratio of 2.2: 1. [Table 1] shows the age distribution of the patients collected. Most of the patients were aged between 16 and 20 years (33%), followed by 6-10 years (19.0%) and 11-15 years (16.7%). Patients aged 21-30 years and 41-50 years constituted 7.2%, whereas 9.6% of patients were above 50 years. The ages of pediatric patients ranged between 3 and 17 years, whereas that of adult patients varied between 18 and 86 years. The mean age in pediatric and adult patients was 7.1 years and 31.3 years, respectively.
The most common complaints among all patients were generalized weakness (71.4% cases) and fever (61.9% cases), followed by easy fatiguability (42.8%), breathlessness (30.9%), and progressive paleness (19.0%). Purpura, melena, or per rectal bleeding were the most common bleeding manifestations.
Severe pallor was noted in 16 (38.1%) patients, followed by moderate pallor in 14 (33.3%) and mild pallor in 28.6% of cases. Purpuric spots and lymphadenopathy were observed in 11 (26.2%) and six (14.3%) patients, respectively. No organomegaly was noted in any patient. The comparison of clinical findings of the pediatric and adult patients is depicted in [Table 2].
Pediatric patients presented mostly with fever and generalized weakness, whereas weakness was the predominant symptom in adults. Progressive pallor was more common in children, whereas breathlessness was more common in adults. There was no significant difference in the distribution of cases, both pediatric and adult, presenting with mild, moderate, and severe pallor.
Most of the patients, around 78.6%, presented with severe anemia. Total red blood cell counts were mostly (52.4% of patients) between one and three millions/μl, whereas in 26.2% of patients it was below one million/μl. Most patients (92.8%) presented with total leukocyte counts between 1000 and 4000/μl, whereas rest of the patients had normal leukocyte counts. Most patients had mild (50% of patients) and moderate (38.1% of patients) absolute neutropenia. Platelet count was less than one lakh/μl in all patients. Among them, severe thrombocytopenia was observed in 52.4% of patients. Hematological profile of the patients is presented in [Table 3].
Hemoglobin values varied more widely in pediatric patients than in adults. The occurrence of severe anemia was greater in pediatric patients (81.8%) compared with adults (70%). The total leukocyte count was less variable in pediatric patients and was more toward the lower side of normal range, with a mean value of 2024/μl. The total leukocyte count varied more in adults, with a mean of 2814/μl. The absolute neutrophil count was much lower in pediatric patients, with a mean of 324/μl, whereas the value was 628/μl in adults. There was no significant variation in the distribution of platelet counts among pediatric and adult age groups ([Table 3]).
The occurrence of nonsevere and severe diseases was almost the same in children. Only around 14% of pediatric patients had very severe disease. Nonsevere disease was common in adults, and only 10% of patients had very severe disease ([Table 4]).
Patients presenting with mild pallor had hemoglobin values between 3.1 and 12.1 g/dl, with mean and median values of 6.3 and 5.4 g/dl, respectively. Those with moderate pallor had hemoglobin values ranging from 2.9 to 8.8 g/dl, with mean and median values being 4.4 and 5.1 g/dl, respectively. Patients with severe pallor had hemoglobin values ranging between 1.6 and 5.9 g/dl, with the mean and median being 3.1 and 2.3 g/dl, respectively ([Table 5]).
Patients presenting with fever had total leukocyte count ranging from 1100/μl to 7200/μl, with mean and median values around 2000/μl, whereas absolute neutrophil count in those patients ranged between 24/μl and 3312/μl, with mean and median values close to 400/μl. Most of the patients having fever had absolute neutrophil count between 200 and 500/μl (46.15% of patients with fever) and more than 50/μl in 34.62% of patients. Severe neutropenia was observed in 19.23% of patients with fever ([Table 6]).
|Table 6 Correlation between fever and leukocyte count and absolute neutrophil count|
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Patients having minute bleeding had platelet count between 11 000/μl and 71 000/μl, with mean and median values being around 23 000/μl and 29 000/μl, respectively. Patients presenting with gross bleeding had platelet count ranging from 4000/μl to 35 000/μl, with mean and median values around 16 000/μl and 20 000/μl, respectively ([Table 7]).
|Table 7 Correlation between hemorrhagic manifestations and platelet count|
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During the procedures for collection of bone marrow samples, no serious complication such as bleeding, needle break, and local anesthetic reaction were noted, except for local hematoma formation in seven (21.8%) patients. In only two (6.2%) patients, bone marrow aspiration procedures were met with dry tap. Patients were followed up for up to 2 weeks after the procedures, and no complication was encountered. The findings of bone marrow aspiration and imprint in relation to their corresponding biopsy are presented in [Table 8] and [Table 9].
The findings of bone marrow aspirations [Figure 1] varied considerably compared with their corresponding biopsies. An overall 28.6% of bone marrow aspiration samples were nondiagnostic because of inadequate cellularity or blood tap. In two patients, aspirate smears showed a few normocellular particles in the background of predominantly hypocellular particles. Rest of the successful aspirates (n = 31) correlated well with the hypocellular bone marrow biopsies [Figure 3].
The cell density of bone marrow imprint smears [Figure 2] correlated well with the cellularity of the bone marrow biopsy in 78.6% of patients. Imprint smears were uninformative in six (14.3%) patients, as the smears were acellular. In three (7.1%) patients, the cell density of imprint smears was more than could be expected from aplastic marrow.
Bone marrow aspirate smears were not helpful in 28.6% of patients due to dry tap, blood tap, or aparticulate smears. In two patients, aspirate smears showed a few normocellular particles in the background of predominantly hypocellular particles. In those two cases, imprint smears were fairly cellular. Rest of the successful aspirates (n = 31) correlated well with the hypocellular bone marrow biopsies. In contrast, imprint smears had findings similar to aspirate smears and correlated with the hypocellular biopsy specimens in 73.4% of patients.
All of the bone marrow core biopsy specimens floated in the decalcifying solution after decalcification [Figure 7], and this finding correlated with hypocellular bone marrow on biopsy examination in all patients.
A total of 24 whole-blood samples were collected from patients and cultured. The culture failed in nine cases and did not yield an adequate cell pellet during harvesting. The culture was successful in 15 cases, and no significant cytogenetic abnormality was noted among them [Figure 6], except for single complex aberration and single chromosome break in one case each.
An overall 38.1% of patients could not be followed up subsequently because of referral or the patient not attending follow-up consultation. In all, 54.8% of patients were alive while writing this paper and death was documented in three (7.1%) patients.
| Discussion|| |
A total of 42 patients were diagnosed with aplastic anemia, with a male to female ratio of 2.2 : 1 and a pediatric to adult ratio of 1.1: 1. Most of the patients were aged between 6 and 20 years. The mean age of male patients was 16 years, and in female patients it was 21 years.
Most of the previous studies , reported a slightly higher incidence of aplastic anemia in female population, but in Asian studies , and in our present study the incidence was considerably higher in male patients.
Most of the patients presented with symptoms of generalized weakness, fever, easy fatiguability, and breathlessness in descending order of frequency. There was little difference in the distribution of patients having mild, moderate, and severe pallor. Around one-fourth of the patients presented with purpura. Organomegaly was observed in no case. Our observation was more or less similar to that of previous studies  .
Most pediatric cases, in our study, presented with fever and generalized weakness, whereas weakness was the predominant symptom in adults. Progressive pallor was more common in children, whereas breathlessness and gross bleeding in the form of hematuria, epistaxis, or melena were more common in adults.
Hemoglobin levels varied widely in children compared with adults. The occurrence of severe anemia was greater in pediatric patients (81.8%) than in adults (70%). The total leukocyte count in children was less variable and more toward the lower side of normal range, with a mean value of around 2024/μl, whereas the values were more variable in adults with the mean being 2814/μl. There was no significant variation in the distribution of platelet counts. The absolute neutrophil counts were much lower in pediatric patients, with a mean of 324/μl, whereas the value was 628/μl in adults.
The range of hemoglobin values and total leukocyte count were wider in the present study compared with previous studies. The mean values of platelet count and absolute neutrophil count, in present study, were intermediate between the other two studies.
Biopsy specimens could be obtained in all the 42 finally diagnosed patients of aplastic anemia; however, despite repeated attempts of bone marrow aspirations, seven (16.6%) and two (6.2%) patients were met with blood tap and dry tap, respectively. No adverse event such as bleeding, needle break, local anesthetic reaction, or death was encountered. Only local hematoma formation was observed in seven (21.3%) patients. Patients were followed up for up to 2 weeks after the procedures, and no complication such as infection was documented. In a study conducted by Barbara J. Bain (2003) on 54 890 biopsy samples, adverse events were seen in 26 (0.047%) cases, mostly comprising hemorrhage in 14 (0.028%), needle-related incidents in seven (0.014%), and infection in three (0.006%) cases  . None of the patients having hemorrhage suffered from aplastic anemia. Six of those 14 cases with hemorrhage needed blood transfusion, and one patient ultimately died of bleeding. Our observations are in agreement with that of Barbara J. Bain in that there was no incidence of bleeding in aplastic anemia.
In 4.8% of cases, there were a few normocellular particles admixed with predominantly hypocellular particles. Rest of the successful aspiration samples were hypocellular (66.7%).
In most of the cases (73.8%), bone marrow imprint smears showed evidence of marrow aplasia/hypoplasia and suggested the diagnosis of aplastic anemia, whereas in 26.2% of cases the imprint smears were not helpful. Combining the findings of aspirations and imprints altogether, aplastic anemia was suggested by both findings in 71.4% of cases, whereas in 23.8% of cases the imprint smears were more helpful for diagnosis compared with aspiration smears.
The bone marrow biopsy findings were compatible with the diagnosis of aplastic anemia in all the patients. The findings of bone marrow imprints and biopsies were comparable in 76.2% of cases; however, the imprint smears were uninformative in 16.7% of cases because of acellularity and in 7.1% of cases cellularity of imprint smears was than could be expected in aplastic anemia.
Our observations were comparable with those in a study conducted by Lu XG et al.,  who graded cellularity of bone marrow imprint smears into four grades: distinct decrease, extreme decrease, distinct increase, and extreme increase, and reported that the cellularity was significantly higher in imprints than those in aspirate smears, but there were no significant differences between marrow imprints and biopsy sections in terms of cellularity.
From our observations, we found that bone marrow aspiration smears and imprint smears differ considerably in their positive predictivity in diagnosing aplastic anemia. Marrow aspirations can be unsuccessful in marrow failure resulting in dry tap or blood tap. Acellular or paucicellular aspirations can occur in marrow aplasia or packed marrow in leukemic marrow, myelodysplasia, myelofibrosis, etc. Hypocellular marrow aspirations can be met in aplastic anemia, hypocellular myelodysplastic syndrome, myelofibrosis, and several other causes; hence, mere hypocellular particles in bone marrow aspiration smears cannot be taken as diagnostic.
In two cases, quite a few (around 10%) normocellular particles were seen in the background of predominantly hypocellular particles. This can be explained by aspiration from a 'hot spot' [Figure 4], or an area of bone marrow in which hematopoiesis is not affected to its full extent. In such cases of variable cellularity, it was difficult to describe the overall cellularity of the bone marrow depending upon aspiration smears.
Bone marrow imprints, in contrast, were far more dependable as they were representatives of overall cellular density of the marrow core biopsies obtained from two different sites. The chances of getting acellular or paucicellular imprints are less, as they are prepared by touching glass slides directly with the marrow cores. Their cellular density was, in most of the cases, suggestive of marrow suppression; however, in 16.7% of cases the cellular density of imprint smears was more than could be expected from a case of aplastic anemia.
Bone marrow biopsy was the tool that gave us the final and accurate details of marrow cellularity and proportions of each individual cellular element. It nullified the bias created by normocellular particles in marrow aspiration smears. All the problems of assessing marrow cellularity arising from blood tap and paucicellular smears were solved by biopsies. Furthermore, assessment of marrow fibrosis with reticulin stain helps to distinguish aplastic anemia from hypocellular myelodysplastic syndrome and myelofibrosis , . All cases were eventually diagnosed by means of bone marrow biopsy findings, combining with findings of peripheral blood, bone marrow aspirations, and marrow imprints. Finally, we can conclude that bone marrow biopsy can be taken as the gold standard for diagnosing marrow suppression in aplastic anemia; however, cellular morphology interpretation is better in aspiration and imprint smears compared with biopsies.
The floatation of the bone marrow core biopsy specimens after proper decalcification, as was noted in all the cases in our study, may turn out as a reliable tool for assessing the overall marrow cellularity. Reduced cellular density of the bone marrow and replacement of the hematopoietic cellular elements by adipocytes might be the cause of this finding. The combination of evidences obtained from clinical details, peripheral blood pancytopenias, hypocellular marrow particles in aspirate smears, and poorly cellular imprint smears with or without adipocytosis, together with the floatation of core biopsy specimen can be accepted as fast and dependable findings for diagnosing aplastic anemia, without waiting for the biopsy report, which takes a longer period of processing. This may help the clinician to initiate a prompt treatment in critically ill patients. However, to derive a statistically significant conclusion, larger studies involving aplastic anemia and other hematological disorders are required.
Five patients could not be contacted because of insufficient or incorrect personal contact details, four patients did not attend follow-up consultations, three patients died within a few days after collection of bone marrow samples, and six patients were unwilling to provide their blood samples for cytogenetic studies.
A total of 24 samples from patients of aplastic anemia and eight samples from age-matched unrelated healthy volunteers were collected in the Department of Pathology, Institute of Medical Sciences, Banaras Hindu University, and were immediately transported to the Centre for Cytogenetic Disorders, Banaras Hindu University for cytogenetic work up.
Whole-blood lymphocyte culture failed in nine cases as they could not yield an adequate cell pellet during harvesting. A cause of severe leucopenia can be attributed to this, as was encountered in all those nine cases. For better yield of metaphase spread, bone marrow aspirate sample, in place of peripheral blood sample, may be used for lymphocyte culture.
The cultures were successful in 15 cases and were evaluated for chromosomal abnormalities with or without the effect of MMC. The observations were compared with the corresponding age-matched control samples.
No cytogenetic abnormality was observed in any of those 15 cases, except for single insignificant chromosomal gap and insignificant complex chromosomal aberration in one case each. Our results are comparable to the study conducted by Yarnal and Basu  , who also encountered failure in yielding metaphase in 16 cases among a total number of 30 cases of aplastic anemia, and in 11 cases normal karyotype was detected. Trisomy 6, trisomy 8, and loss of chromosome 7 were detected in one case each.
Increased chromosomal breakage upon exposure to MMC (50 mmol/l dose) was not reported in any case. We have analyzed chromosomal breakage on exposure to single dose of MMC. It is recommended to analyze with multiple increasing doses (e.g. 50, 150, 300 mmol/l etc.) of MMC to diagnose Fanconi anemia.
No significant statistical conclusion can be drawn out from our study on cytogenetic profile of aplastic anemia because of small sample size; hence, larger studies are required to confirm and assess the magnitude of chromosomal abnormality in patients with aplastic anemia.
| Conclusion|| |
Aplastic anemia, a potentially fatal disease, can occur in any age group, although mostly affects children and adolescents. It presents with clinical symptoms and signs, resulting from suppression of hematopoiesis - for example, generalized weakness, fatigue, paleness of the body, fever, and purpuric spots.
There is no significant difference in the clinical presentation among the pediatric and adult aplastic anemia patients. The levels of hemoglobin concentration and platelet count also did not show considerable differences among the two populations; however, total leukocyte count and absolute neutrophil count were significantly low in pediatric patients. The degree of pallor correlated well with the hemoglobin concentrations. Fever was more common with low absolute neutrophil count. The severity of bleeding manifestations correlated with the severity of thrombocytopenia.
Although bone marrow aspiration helps in diagnosing marrow aplasia or hypoplasia, the confirmative diagnosis rests upon bone marrow biopsy examination, as it gives more precise information about marrow cellularity and also excludes several other causes of hypocellular marrow aspirate. Bone marrow biopsy is indeed necessary to differentiate aplastic anemia from hypocellular myelodysplastic syndrome and myelofibrosis.
The observation of floatation of marrow core biopsy specimens after proper decalcification can serve as a reliable tool for assessment of overall marrow cellularity; however, larger studies are required for validation of this finding.
Cytogenetic work up could be carried out in small number of cases in our study and no chromosomal abnormality was documented in any of them; however, an extensive study on cytogenetic profile in patients with aplastic anemia is strongly recommended for any statistically significant conclusive opinion ([Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]).
| Acknowledgements|| |
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]