Aplastic anaemia (AA) is a rare disease of bone marrow (BM) failure characterized by peripheral pancytopenia and marrow hypo-cellularity in the absence of an abnormal infiltrate and no increase in reticulin . The disease may be categorized into two groups i.e. inherited and acquired. The foremost types of inherited AA (IAA) are Fanconi anaemia, Dyskeratosis congenita, Shwachman Diamond syndrome, Diamond Blackfan anaemia, etc... An additional category of AA is acquired aplastic anaemia (AAA), which was thought to be a result from atrophy or chemical injury of primitive marrow hematopoietic stem cells (HSCs) . But the unexpected recovery of marrow recipients after having immunosuppressive conditioning therapy upraised the viewpoint that the disease may not be intrinsic to primitive HSCs, nonetheless a consequence of the suppression of the same by immune-mediated destruction, remarkably by autoreactive T lymphocytes . The potential aetiological factors of AAA are drugs, toxic chemicals, viruses, paroxysmal nocturnal hemoglobinuria (PNH), autoimmune/connective tissue disorders, thymoma, thymic carcinoma, pregnancy, radiation, cytotoxic drug therapy, idiosyncratic reactions, hyper-immunoglobulinemia, Graft vs. Host Disease etc. The occurrence of AAA in the Western countries is about 2 per million per year, whereas it occurs more commonly in the Far East, with a 2 to 3 fold higher prevalence [3,4].
In our country, AA patients are exposed to different known/unknown aetiological agents by their occupation and lifestyle. As well as due to the low socioeconomic background and awareness, the AA patients are having different kinds of addictive substances. But their aetiological contribution to the disease progression has not been evaluated till date. However, in a large number of AA cases, the definite etiological factor is unknown. In those cases AAA is called idiopathic AA.
Investigation about the role of the exposure of some established and not established aetiological factors and addictions in AA patients was the theme of this study.
Materials and methods
Approximately 139518 patients in total, with a different type of haematological disorders, were screened at the Department of Haematology, Nil Ratan Sircar Medical College & Hospital from 2015 - 2018. Diagnosis of AA was made conferring to the guidelines of International Agranulocytosis and Aplastic Anaemia Study Group, 1987 .
At least two of the three criteria with hypocellular bone marrow must be present to define AA which is, (1) haemoglobin<100 g/L,
(2) platelet count <50 X 109/L, (3) neutrophil count <1.5 X 109/L. Bone marrow aspiration and biopsy (BMA & BMB) examination was done of 4032 pancytopenia patients to endorse AA. BMA & BMB is an invasive technique it cannot be performed repeatedly. Considering all pancytopenia cases as a potential case of AA, we took BM and PVB samples. Later, only those cases were selected, who reports for the presence of hypo-plastic anaemia or hypocellular bone marrow.
Stress cytogenetics/ chromosomal breakage test was done (<50 years) using Mitomycin C to exclude inherited BM failure syndrome i.e. Fanconi anaemia (mostly found) .
Ethical clearances were obtained both from the Ethics Committee of Vivekananda Institute of Medical Sciences and Nil Ratan Sircar Medical College & Hospital.
Questionnaire and consent form administration
Patients and control cases were thoroughly informed about the research work. Written consent was taken before the collection of samples. A detailed questionnaire was administered to record their data. Different types of exposures like pesticide, toxic chemicals, drugs, occupation, drinking water, addictions etc. were included in the questionnaire.
Although addictions have been found to be associated with several life-threatening diseases, the role of addiction and their implication in the marrow aplasia is not studied before. Keeping this fact in mind, some widely used addictive substances like tobacco (leaf, chewing, smoking), alcohol, betel leaf/quid, arecha nut etc... The addicted and exposed AA patients were in a practice of the same cumulatively at least for five years, before and after the disease diagnosis.
Collection and analysis of arsenic from the hair sample 170 hair samples of AA patients and 170 controls were collected. The hair samples were commercially outsourced for arsenic estimation. The samples were thoroughly washed with distilled water followed by de-ionized water and finally by acetone in an ultrasonic bath. The washed samples were placed in glass beakers individually and allowed to dry at 500C overnight in a drying oven. Total arsenic was determined after nitric acid digestion in a Teflon bomb at about 1000C. A mixture of nitric acid and hydrogen peroxide (30% (v/v)) were also used for sample digestion in a glass beaker placed on a hot plate at 80-900C for 2-3 days until the solution was clear. The filtered solution was used for total arsenic determination by the method of flow injection hydride generation atomic absorption spectrometry.
Odds ratio (OR) was used to signify the odds that an outcome will occur given a specific exposure, in comparison to the odds of the outcome occurring in the absence of that same exposure. OR=1 means exposure does not affect odds of the outcome; OR>1 means exposure associated with higher odds of the outcome, OR<1 means exposure associated with lower odds of the outcome. A confidence interval was used to refer the percentage of all possible samples that can be expected to include the true population parameter. Further, to minimize the interactions or interference among the factors resulting in falsepositive or false-negative results, multiple-factor regression analysis was performed to make the results more statistically potent. Coefficient of Determination (R2) and Correlation Coefficient (r) were also calculated. The Mann-Whitney U test was used to compare the values of arsenic count in AA patients with the controls. The p-values between the exposed and nonexposed groups were calculated by Chi-square method.
Results obtained from the questionnaire
170 diagnosed AA patients participated in this study. The median age was 50 years (n=170). The age range of the patient was 2-85 years. (Figure 1) showed the highest peak of the AA patients at the age of 5- <15 years and at 50-<60 years respectively. Among them 105 (61.76%) were male and 65 (38.23%) were female. Out of these patients, 30% (n=51) came out with very severe AA (VSAA), 52.94% (n=90) came out with severe AA (SAA) and 17.06% (n=29) came out with non-severe AA (NSAA).
Results obtained from the stress cytogenetics study
The age range of the patient was 2-49 years. The median age of the patients was 23 years (n=99). The median age of the patients (+ve) for Stress cytogenetics was 5 years (n=9). Out of which, 69.69% (n=69) were male and 30.30% (n=30) were female. Out of these patients, 41.41% (n=41) came out with VSAA, 45.45% (n=45) came out with SAA, and 13.13% (n=13) came out with NSAA. Nine patients (9.09%) were found to be (+ve) for stress cytogenetics, suggestive for FA. In Stress Cytogenetics (+ve) cases, six out of nine cases (66.66%), history of consanguinity was found.
The percentages of AA patients came from different districts of West Bengal and other states of eastern India In this study, patients were taken from the nodal centre for haematology of Eastern India, which is Nil Ratan Sircar Medical College & Hospital situated in Kolkata. The high percentages of AA patients came from different districts of West Bengal (97.06%) as well as other states (2.94%) of Eastern India. Their exposure to different aetiological factors, occupation, addictions and water consumption are tabulated in table 1.
Results of the aetiological factors obtained from the case and control analysis
After carefully analyzing the data from the questionnaire the number and percentages of individual patients and controls exposed to different types of aetiological (established /non established) (Table 1) is prepared, where their p-value, odds ratio, relative risk and 95% confidence interval (CI) were also summarized. Furthermore, multiple regression analysis was performed to find out the coefficient of determination (R2) [it ranges from 0 - 1] which is one of the key output of regression analysis.
It is understood as the proportion of the variance in the dependent variable that is predictable from the independent variable.
R2 = 0; means that the dependent variable cannot be predicted from the independent variable.
R2 = 1; means the dependent variable can be predicted without error from the independent variable.
R2 between 0 and 1; indicates the extent to which the dependent variable is predictable.
From the calculated coefficient of determination (R2), correlation coefficient (r) .
The equation obtained from the multiple regression analysis is as follows.
y = 5.254794366.10-2 C1 - 5.649505803.10-2 X2 + 3.040525941 Coefficient of Determination: R2 = 0.2051339935 ≈ 0.2 An R2 of 0.2 means that 20 percent of the dependent variable, relative risk, can be predictable from this set of data of case control study. To be more lucid, this set of data is portraying that the risk of developing AA is 20 percent in presence of the exposure of the above mention aetiological agents in this particular population.
Correlation coefficient (r) was calculated (r=0.4523). This was highly significant in the context of this scenario.
Study of arsenic exposure
The patients and controls are divided into three groups based on arsenic count i.e. within the safe limit (WSL) [0.08-0.25mg/kg], above the safe limit (ASL), above the toxicity level (ATL) [1.0mg/ kg]. A very high percentage of AA patients exhibited arsenic count above the safe limit and above the toxicity level compared to control. Although a good number of AA patients were in within the safe limit group, sill there is a significant difference in the mean +/- standard deviation of the arsenic count in the WSL group and control. The mean+/- standard deviation of the arsenic count in the hair of AA patients and control cases is shown in (Figure 2).
Results of statistical analysis of the environmental and lifestyle factors in AA patients participated in this study a) The study of pesticide exposure showed the sequence of OR (high- low) is repellents > rodenticides >insecticide> piscicides > fungicide> bactericide> non-exposed to the pesticide. A significant p-value (<0.0001) was found between the exposed and non- exposed group.
b) The study of chemical exposure showed the sequence of OR (high- low) is benzene = petrol- diesel smoke>TNT= gold / silver salt> lead paint, paint varnish> coal-tar chemical >chlorinated hydrocarbon> rotenone (fishing chemical> DDT> others>stoddard solvent> non-exposed to chemicals.
A significant p-value (0.000089) was found between the chemical exposed and non-exposed group. AA patients with radiation exposure did not participate in this study.
c) The study of drug history showed the sequence of OR (highlow) is chloramphenicol > NSAID = others > D-penicillin = acetazolamide = hydantoins > quinacrine = carbamazepine > no history of drug taking. A significant p-value (0.000076) was found between the users and non-users' group.
d) The study of occupation showed the sequence of OR (high- low) is farmer> student> driver> factory worker> housewife> others > unemployed> service. A significant p-value (0.002354) was found between the employed and non- employed group.
e) The study of drinking water showed the sequence of OR (high- low) is shallow tube well> well> pond> deep tube well> packaged water> boiled water> corporation line. A significant p-value (<0.0001) was found between the safe and unsafe water consuming group.
f) The study of addiction showed the sequence of OR (high- low) is processed chewing tobacco> smoking> alcohol> others> tobacco leaf> betel leaf/quid> non-addicted. A significant p-value (<0.0001) was found between the addicted and nonaddicted group.
g) The study of arsenic count showed the sequence of OR (highlow) is arsenic count above the toxicity level > arsenic count above the safe limit > arsenic count within the safe limit. A significant p-value (<0.00001) was found between the arsenic count [(above the toxicity level group + above the safe limit group) and arsenic count within the safe limit group]. In every case, there were some AA patients who did not report any exposure. In the category of the source of the drinking water, the p-value was calculated between safe water (packaged water, boiled water, corporation line) and unsafe water (pond, well, hallow tubewell, deep tubewell). In the category of occupation, the p-value was calculated between employed (farmer, factory worker, driver, student, housewife, service, others) and unemployed.
Aplastic anaemia (AA) is defined as pancytopenia with hypocellular bone marrow in the absence of abnormal infiltrate, malignant cells, and fibrosis with no increase in reticulin . Many western studies have already focused on the various aspects of AA. But there is very little documentation on AA from India. In eastern India, virtually the research is at the budding phase and this study is the first documentation from eastern India focusing on the aetiological factors in AA patients.
Existing documentation indicates that AA is associated with a lower socioeconomic profile and environmental exposure to several toxic substances among the Pakistani population . Individuals who were exposed to pesticides were more likely to be diagnosed with AA [8-10]. Additionally, even the general inhabitant, living in areas with extensive agricultural operations,has high exposure to pesticides . An investigation on the role of occupational and environmental exposures on the aetiology of acquired SAA was also performed in the UK . Among the various aetiological factors associated with AA, pesticides [13-15], arsenic [16-18] and chemicals like benzene  have been found to be strongly linked with an increased susceptibility of individuals to develop AA. In a study, BM spicules of Megaloblastic anaemia patients were examined by electron-probe analysis to demonstrate the presence of arsenic and they showed arsenic in BM spicules .
A study focused on the role of organochlorine compounds, Parvovirus B19, Hepatitis viruses B and C and HIV I and II in the causation of AA in children . In India, arsenic is associated with carcinoma of lungs, liver, kidney and bladder . A case study has been reported from the Mumbai, focused on the AA due to heavy metal intoxication .
A single study had been conducted to find the association of implicative factors of this disease in the population of West Bengal. In West Bengal, pesticide-induced alterations in marrow physiology and the depletion of the stem and stromal progenitor population have been reported . It is already established that West Bengal is the most arsenic affected region beside Bangladesh in the world . It is necessary to have a look at the arsenic level in the AA patients of West Bengal.
A lower socioeconomic status is a risk factor for AA [24, 25]. A study has suggested and emphasized on the fact that besides host genetics, several other hemotoxic factors may contribute to an environmental aetiology of AA . Evidence related to a higher association of hepatitis infection with AA is also present [27-29]. The environmental toxic exposures, pesticides were found to be strongly associated with the AA disease occurrence [15-30]. Studies report that occupational exposure to pesticides among farmers is strongly associated with all hematopoietic cancers .
We took the help of the questionnaire to evaluate the possible environmental and lifestyle factors. Some proven and some nonestablished etiological agents are included in the questionnaire.
We have discussed the percentages of AA patient's exposed to different environmental factors in the results section. It was observed that most patients came from Kolkata, followed by South 24 Parganas and Murshidabad. But this may happen because we have collected samples only from Nil Ratan Sircar Medical College & Hospital. It is the only nodal centre of Haematology in eastern India. These districts are very much adjacent to the aforesaid hospital. A small number of patients were participated from the other states of eastern India, because of low awareness and knowledge about the study programme and the institutions. The 83% of AA patients belong to the very low socio-economic status being the average monthly income of the family was less than rupees 2000/-. Lack of affordability makes the whole procedure critical to the patients of haematological disorders.
Various type of occupation were found in the patients' population. Common occupations were observed to be farmer, factory worker, housewife, student, driver, government or nongovernment service and others. Farmers are having potential exposure to insecticide and pesticides. Relation of AA with pesticides was already documented . Some of the farmers were also using the organophosphate as a pesticide which is a potential aetiological agent of AA. Some frequently used chemicals by the AA patients are gold/silver salt, lead associated paint, paint varnish, coal-tar etc...The occupations of AA patients were also discussed. Some patients reported their occupation to be fishing and for the same, they are exposed to certain fishing related chemicals like sodium cyanide, PCBs, Rotenone, mercury chemical etc. Needless to mention some patients have reported their occupation as driving. They were being constantly exposed to the smoke of petrol and diesel due to the increased level of pollution. Few reported documentations on fishing chemicals and petrol/ diesel (both in smoke and liquid form) are present .
But their relation to the suppression of hematopoietic stem cell compartment is still unclear. A high percentage of patients were found to be not exposed to any known aetiological factors related to AA. This observation also a support the conventional concept of AA, as in 50% cases of AA the primary aetiology is unknown and is called idiopathic aplastic anaemia .
Consumption of different addictive substances has been observed in patients of AA. Although no concrete reported data are found which emphasized on the pathological role of addictive substances in AA. Use of tobacco in raw (tobacco leaf), chewable (khaini, gutkha, zarda), smoking (cigarette, bidi) and powder (snuff) form were found in the patients of AA. Betel leaf and betel quid was the maximum used addictive substance. Areca nut solely or with betel leaf was also consumed at a very high percentage. Alcohol is a proven factor related to pancytopenia with cellular BM . AA considerable percentage (12.94%) of patients showed alcohol consumption. Patients participated in this study did not report for Non-A, B, C, D, E hepatitis virus, EBV, CMV, HIV infections. A more in-depth study on a large population can reveal the actual effects of the above mentioned aetiological agents.
We focused on the different types of sources of water, consumed by the AA patients for the last five years. A very small population of our society has access to purified or boiled water. The mostly used water source is shallow tube well. Source of water consumption is an important factor as groundwater is often contaminated arsenic, fluoride, pathogens, nitrate, organic compounds, metals (lead, cadmium and chromium), organohalides, petroleum hydrocarbons etc... In this study, a considerable percentage of AA patients showed arsenic count above the safe limit compared with control. Again, a high percentage of AA patients showed arsenic count above the toxicity level. Virtually only two control sample reports for the arsenic count above the toxicity level.
There are many pieces of evidence behind environmental factors can induce human cancers, telomere dysfunction, neurodegenerative diseases [34-37]. Keeping these studies in mind the data of environmental and lifestyle exposure of Fanconi anaemia patients were also collected. In a precise population, there is a likelihood of having the same nature of environmental and lifestyle factors disclosure in the patents which can lead to the mutation in the gene blamable for marrow failure. In such a consequence the children are at extraordinary risk of getting the disease. However consanguineous marriage can augment the frequency of the disease.
The present study was undertaken with a view to evaluating the role of known and unknown environmental factors and lifestyle factors of AA patients in eastern India. The findings there after were recorded carefully, analyzed and corroborated in reference to the aims and objectives of this study and it is concluded environmental factors and lifestyle factors plays a profound role in the disease progression in AA patients of West Bengal. A considerable percentage of AA patients showed arsenic count above the safe limit and above the toxicity level as compared to control. Thus, it can be envisaged that arsenic toxicity can be a responsive factor in AA as West Bengal is an arsenic prone area. For the confirmation of the presence of arsenic, it's estimation from the BM sample can be performed.
In conclusion, it can be said that probably the addiction, occupational or lifestyle exposure have a role in the disease propagation in AA patients. Of course, different types of exposure and addiction do not have the same propensity in the formation of the disease; neither will they make disease in all individuals. This observation supports the fact that every patient is coming from the different genetic background and their retention capacity and clearance of such toxic antigens are variable. This kind of lifestyle habits should be immediately terminated after the disease diagnosis as it can make the disease more complex. Simultaneously the patients should be counselled for the probable risk. Further studies are needed to come to any definite conclusion.
We are obligated to Swami Nityakamananda, Secretary, Ramakrishna Mission Seva Pratishthan, Vivekananda Institute of Medical Sciences, Kolkata for giving us inventory support and Department of Science of Technology, West Bengal (DSTWB) for giving us financial support (Grant No. 512(Sanc.)/ST/P/ S&T/9G-8/2015). We are also grateful to the Department of Haematology, Nil Ratan Sircar Medical College & Hospital for providing samples ando ther assistance.
Disclosure of conflict of interest