Pancytopenia was not a discrete hematological entity even as late as 1919. The term was used almost synonymously for aplastic anaemia, it being the major cause of pancytopenia in the western countries[1]. Aplastic anaemia (AA) is a lifethreatening bone marrow failure disorder, if untreated, is associated with very high mortality. The incidence of AA is higher in Asia than in the West. It appears to be 2 to 3-fold more common in Asia than in Europe[2]. The precise incidence of AA in India is not known due to lack of epidemiological study. However, in hospital based study, it is known that 20-40% of pancytopenic patients are diagnosed as AA in referral centres[3]. In an epidemiological study in children in and around Lucknow, UP, showed the annual incidence of aplastic anaemia is around 6.8 cases per million of population per year[4]. Peripheral cytopenia is defined as reduction in either of the cellular elements of the blood, i.e. red blood cells, white blood cells or platelets. Bicytopenia is the reduction of any of the two cell lines and pancytopenia is reduction of all the three (haemoglobin<10g/dl, absolute neutrophil count 1.5 x 109/l and platelet count <100 x 109/L )[5]. The etiology of bicytopenia and pancytopenia varies ranging from transient marrow viral suppression to marrow infiltration by life threatening malignancies. Pancytopenia can result from a failure of production of hematopoietic progenitors, their destruction, or replacement of the bone marrow by tumor or fibrosis. Although selective cytopenias are important clinical entities, pancytopenia is a loss of all marrow elements. Pancytopenia can be constitutional, arising as a consequence of an inherited genetic defect affecting hematopoietic progenitors, or can be acquired as a consequence of either direct destruction of progenitors, immune mediated damage to either haematopoietic progenitors or their nurturing microenvironment, or suppression of or crowding out of progenitors by tumor cells or fibrosis.

Although Fanconi anemia is the best-recognized constitutional pancytopenia, a number of other infrequent genetic disorders have also been implicated. These genetic syndromes include various modes of inheritance and may be associated with a number of congenital abnormalities, especially of the bones, kidneys, and heart. Because the hematologic manifestations of the congenital pancytopenias may not become manifested until the first years to even decades of life, a genetic predisposition to bone marrow failure should be considered in all cases of aplastic anemia. These disorders can be autosomal recessive (e. g., Fanconi anemia, dyskeratosis congenita), X linked, or autosomal dominant (e. g., dyskeratosis congenita). Several of these genetic disorders may present initially with a single cytopenia and progress to pancytopenia (Swaschman- Diamond syndrome, amegakaryocytic thrombocytopenia, reticular dysgenesis). In addition, a number of inheritable familial marrow dysfunction syndromes have been associated with pancytopenia (which can also be autosomal recessive, autosomal dominant, or X linked), and aplastic anemia also occurs in association with other genetic disorders. Thus, pancytopenia can be either the primaiy disease manifestation or can emerge as a rare complication during the course of another illness. Because of the chromosomal fragility or defective repair mechanisms that may be associated, several of these disorders can also be complicated by cancer or other organ dysfunction(s)[6]  Most common nonmalignant cause of acquired pancytopenia is aplastic anemia followed by megaloblastic anemia. Among the malignant causes acute leukemia is most common. Pancytopenia caused by marrow replacement is seen to occur in leukemia. Pancytopenias can be caused by various drugs-chemotherapeutic agents, antiepileptics (hydantoin, carbamazepine) NSAIDS (phenylbutazone, ibuprofen, diclofenac. Prolonged cytopenias can occur after infection with many of the hepatitis viruses, Epstein Barr Virus, Cytomegalovirus. Pancytopenia may occur after a single high dose of radiation[7]. Non malignant conditions are Immune Thrombocytopenic Purpurc, megaloblastic anemia, marrow hypocellularity and visceral leishmaniasis. Commonest malignant condition associated with bicytopenia is leukaemia, acute lymphoblastic leukemia being commoner.

Pancytopenia usually presents with symptoms of bone marrow failure such as pallor, dyspnea, bleeding, bruising and increased tendency to infections[8]. The main presenting features swith pancytopenia are fever and pallor. Other common symptoms are petechial rash, bleeding and bone pains. Dyspnea on exertion, easy bruising, epistaxis, gingival bleeding and headache may be the presenting features[7]. Oral infections in the form of gingivitis, tonsillitis and pharyngitis may be found[9]. Pancytopenia results in increased risk of fatigue, cardiac failure and infection. On clinical examination hepatomegaly and splenomegaly may be found.

Careful examination of peripheral blood smear for RBC, leukocyte and platelet morphology is important. A reticulocyte count should be done to assess erythropoietic activity. Bone marrow examination should include bone marrow aspiration along with biopsy wherever indicated. Marrow should be carefully examined for morphology and cellularity. Bone marrow examination is a simple and safe invasive procedure, which causes a modest discomfort and can be performed easily. Its greatest utility is for investigating and it is an important diagnostic modality for evaluating the causes of pancytopenia[10]. In pancytopenia, the marrow can be hypocellular or hypercellular. The hypoplastic marrow, which occurs in 2% of pediatric ALL patients, may be misdiagnosed as aplastic anemia. Studies done have shown leukemia to be the second most common cause of pancytopenia in pediatric patients, marginally behind aplastic anemia[11].

In India, the causes of pancytopenia are not well defined, so the present study has been undertaken to evaluate the various causes and to correlate the peripheral blood findings with bone marrow aspirate [12, 13]. Thereby, this data would help in planning the diagnostic and therapeutic approach in patients with pancytopenia. Treatment and prognosis of patients with pancytopenia are governed by the cause and severity of the underlying disease[12].

The present study was conducted in the haematology section of Department of Pathology of Jawaharlal Nehru Medical College, Sawangi (M). The duration of the study was two years from July 2014 to august 2016. It was a prospective analytical study. The study was conducted on total 53 patients attending outpatient and inpatient department of AVBRH, tertiary care centre. Informed consent was taken from all patients prior to enrollment in the study.

All the cases irrespective of age and sex with pancytopenia were included in this study. Diagnostic criteria for Pancytopenia are, i) Haemoglobin less than 10 gm/dl. ii) Total leucocyte count <4000/cumm iii) Platelet count less than 1,50,000/cumm. Complete history was taken and physical examination was performed. For haematological study, blood samples were collected in EDTA anticoagulant. Complete blood count was carried out using Automated Haematology Analyser. It is an automated cell counter based on the principle of impedance. Hematological parameters considered in this study were haemoglobin, total leucocyte count, platelet count and red cell mass. Peripheral smear were stained using leishmans stain for all the cases and examined in details. Peripheral smear was used for morphological classification and typing of anemia. Bone marrow aspiration was then carried out under all aseptic precautions and slides were stained with leishmans stain. Cytochemistry was done using Myeloperoxidase stain and periodic acid and Schiff reagent stain were performed where necessary. Significant parameters like aetiology, age, gender, clinical features, hematological parameters, peripheral blood film, bone marrow aspiration findings in different cases of pancytopenia were compared with various studies published in literature. Inclusion criteria: Patients of all age groups and sex presenting with pancytopenia. Exclusion criteria: Pancytopenia due to the effect of chemotherapy, radiotherapy and immunosuppresants.

Required for bone marrow aspiration and staining

• Bone marrow aspiration needle.
• Cotton
• Spirit
• Glass slides
• 10ml syringes
• Needle- 22 or 24 No. gauge
• 2% Lignocaine (Local anaesthetic)
• Stains : Leishman stain, MPO, PAS

Method

Phlebotomy

Venous Blood was withdrawn from an antecubital vein or other visible veins in the forearm under all aseptic precautions by using 10 ml syringe peripheral smear was prepared and stained with leishman stain. Haematological parameters like hemoglobin(hb), total leucocyte count (tlc), platelet(plt), RBC were estimated using sysmex automated cell counter.

Bone marrow Aspiration [38]:

• The patient was placed in lateral decubitus position with top leg flexed and lower leg straight.
• The site was prepared, cleaned with antiseptic, draped exposing only the biopsy area.
• Posterior iliac spine was preferred
• The skin and area down to the periosteum were infiltrated with local anaesthetic (2%Lignocaine).
• Skin incision was made with a small surgical blade, through which the bone marrow aspiration and biopsy needles were inserted.
• Bone marrow aspiration needle, with stylet in place, was inserted through the skin, subcutaneous tissue and cortex of the bone with a slight twisting motion.
• A sudden change in give was noted when marrow cavity was entered.
• Then stylet was removed, the hub was attached to 10 or 20 ml syringe and about 0.2 to 0.5 ml of fluid was aspirated.
• Aspiration needle was then removed and pressure was applied to the site with cotton swabs until bleeding stopped.
• Smears of aspirated material were prepared immediately, dried and stained with leishman stain

Leishman’s Stain:

• Weigh out 0.2 g of the powdered dye, and transfer it to a conical flask of 200–250 ml capacity.
• Add 100 ml of methanol and warm the mixture to 50_C for 15 min, occasionally shaking it
• Allow the flask to cool and filter the solution.
• It is then ready for use, but it will improve on standing.
• Buffered Water
• Make up 50 ml of 66 mmol/l So¨ rensen’s phosphate buffer of the required pH to 1 litre with water at a pH of 6.8

Steps:

• Air dry the film and flood the slide with the stain. After 2 min, add double the volume of water and stain the film for 5–7 min.
• Then wash it in a stream of buffered water until it has acquired a pinkish tinge (up to 2 min).
• After the back of the slide has been wiped clean, set it up right to dry

Myeloperoxidase stain[39]:

• Fix air-dried smears for 30 s in cold Buffered formol acetone
• Rinse thoroughly in gently running tap water and airdry.
• Incubate for 10 min in working substrate solution.
• Thoroughly mix 30 mg3,3-diaminobenzidine DAB in 60 ml buffer, add 120 ml H2O2 and mix well. Counterstain with haematoxylin for 1–5 min, rinse in running tap water and air dry

Periodic acid Schiff [39]:

Steps

1. Fix films for 15 min in methanol.
2. Rinse in gently running tap water and air dry.
3. If required, expose fixed control films to digestionin diastase (100 mg in 100 ml of 0.9 g/l NaCl) for 20–60 min at room temperature.
4. Flood slides with 1% periodic acid for 10 min.
5. Rinse in running tap water for 10 min and air dry.
6. Immerse in Schiff’s reagent for 30 min in a Coplin jar with a lid (the Schiff’sreagent can be returned to thestock bottle after use).
7. Rinse in running tap water for 10 min and air dry.
8. Counterstain in aqueous haematoxylin for 5–10 min

The present study is an analytical type and prospective study conducted on 53 cases which were diagnosed as pancytopenia on peripheral smear and was confirmed on bone marrow aspiration was carried out in the Department of Pathology, Jawaharlal Nehru Medical College, Sawangi (Meghe) Wardha, Maharashtra. The duration of study period was from 1^st august 2014 to 31st July 2016.

Age group of patients in the present study ranging from 1 year to 70 years with a Mean of 33.90. Highest number of patients i.e. 15 (28.30%) belong to the age group of 21-30 years followed by 10 patients (18.87%) in the age group of 31-40 years followed by 9 (16.98%) belong to the age group of 11-20 years, 8 (15.09%) belong to the age group of 41-50 years,5(9.43%) of age group of 61 -70 years,4(7.55%) of age group of 51-60 years, lowest were 2(3.77%) patients of age group of 1-10 years.

In the present study, 25 were male patients (47.17%) and 28(52.83%) were females. In present study in females, pancytopenia was common in the 21-30 years age group (10 cases) followed by age groups 31-40 years and 41-50years (5 cases in both groups) 11-20 years (3 cases) 51-60 and 61-70(2 cases each) followed by1-10 years (1case). Pancytopenia was common in the age group of 11-20 years (6 cases) followed by the age groups 21-30 years(5 cases), 31-40 years (5cases) and 61- 70 years(3 cases) 51-60 years(2 cases)followed by 1-10 years(1 case). Male: Female ratio was 1:1.12. Hence pancytopenia was more common in females in the present study.

In the present study most common presentation was of generalized weakness i.e 36 (67.9%) followed by fever 33(62.26%) followed by bleeding6 (11.32%), and edema in 3(5.66%) cases. Pallor was the most common finding was present in all patients i.e 53(100%) followed by splenomegaly i.e in 32cases (60.4%) followed by hepatomegaly in 18 cases (33.96%) followed by lymphadenopathy i.e 12 cases (22.64%), the minimum patients presented with petechiae i.e 9 cases (16.98%).

Table 1: Total no. of diagnosed cases of pancytopenia

The commonest finding in the present study was megaloblastic anaemia 28(52.8%).followed by acute leukemia 11(20.8%) and normoblastic erythroid hyperplasia 6(11.3%), aplastic anaemia 5(9.4%), other causes were mixed nutritional anaemia, multiple myeloma and Non hodkins lymphoma all had 1(1.9%) case each. (As shown in table 1)

Table 2: Peripheral Blood findings in 53 cases of Pancytopenia

A detailed peripheral blood smear examination was done on all patients. Megaloblastic anemia had 28 cases out of which anisocytosis was seen in all 28 (100%) patients, followed by hypersegmented polymorphs in 25(89.29%), followed by lymphocytosis in 16(57.14%). followed by circulating erythroblast in 4 (14.29%). There were 11 patients of acute leukemia immature cells were seen in 10(90.91%) cases, followed by erythroblast in 5(45.45%), followed by anisocytosis and lymphocytosis in 2 (18.18%), aplastic anemia had total 5 cases out of which lymphocytosis was present in all cases, i.e 5 (100%), followed by anisocytosis i.e 2(20%), followed by immature cells in 1(10%). In mixed nutritional anemia anisocytosis, hypersegmented polymorph, erythroblast and lymphocytosis was present in single case. Anisocytosis and immature (plasma) cells were seen in multiple myeloma. In Non hodgkins lymphoma anisocytosis and lymphocytosis was present in a single case. (As shown in table 2)

Table 3: Mean Peripheral Blood Indices

The mean Hb was highest in Non Hodkins Lymphoma followed by acute leukemia, followed by megaloblastic anemia, followed by multiple myeloma, followed by normoblastic erythroid hyperplasia, followed by aplastic anemia and it was lowest in mixed nutritional anemia. The mean Tlc was highest in multiple myeloma i.e 3200, followed by Acute leukemia, followed by Normoblastic erythroid hyperplasia, followed by megaloblastic anaemia, followed by aplastic anaemia, lowest count was seen in mixed nutritional anaemia. (As shown in table 3) Mean platelet count was highest i.e 55000 in Non hodkins lymphoma followed by Multiple myeloma i.e 49000 followed by megaloblastic anaemia i.e 47071.43±16197.68, followed by Aplastic anaemia i.e 44600.00±7231.87. It was lowest in Mixed nutrition anaemia i.e 32000. Out of 53 cases, Hypercellular marrow was observed in 42 cases (79.24%) followed by normocellular 6(11.32%) and hypocellular marrow in 5 cases (9.43%) respectively. (As shown in table 3)

Out of total 28 cases of megaloblastic anemia 32.08 %( 17/28) were females and 20.75 %( 11/28) were males. The second commonest presentation was of acute leukemia, out of 11 cases 15.09%(8/11) were males and 5.66%(3/11) were females, in Normoblastic erythroid hyperplasia total 6 cases were reported out of which 7.55%(4/6) were females and 3.77%(2/6)were males. out of 5 cases of aplastic anemia5.66%(3/5) were males and 3.77%(2/5) were females, a single case reported of mixed nutritional anemia which was a male. Non hodkins lymphoma and multiple myeloma were found in female.

Table 4: Bone marrow diagnosis in 53 cases of Pancytopenia

The commonest finding in the present study was megaloblastic anaemia 28(52.8%).followed by acute leukemia 11(20.8%) and normoblastic erythroid hyperplasia 6(11.3%),aplastic anaemia5(9.4%),other causes were mixed nutritional anaemia, multiple myeloma and Non hodkins lymphoma all had 1(1.9%) case each. (as shown in table 4)

Table 5: Bone Marrow Findings in Cases of Pancytopenia.

All the 53 cases were subjected for bone marrow aspiration findings were: (as shown in table 5)

Megaloblastic Anemia: 28(52.8%) patients with pancytopenia were recognized. Cellularity was found to be increased. Erythropoiesis was hyperplastic and megaloblasts were seen. Early erythroid precursors were found to outnumber mature precursors. Dyserythropoiesis was also noted. Giant metamyelocytes and stab forms ranged from few to fair in number.

Aplastic Anemia: 11(20.8%) of the pancytopenic patients were found to have aplastic anemia. Aspirates were hypocellular with bone marrow fragments being composed largely of fat. M:E ratio ranged from normal to increased Megakaryocytes were found to be diminished. Lymphocytes, mast cells and plasma cells were found to be increased in all cases

Erythroid Hyperplasia: 6(11.35%) of the patients presenting with pancytopenia were found to have erythroid hyperplasia. There was a predominance of erythroid series cells. The reaction varied from normoblastic to mixed reaction. Normoblasts and micronormoblasts were seen in some cases.

Acute Leukemia:- 11(20.8%) of the pancytopenic patients were recognized under this heading were of acute myeloid leukemia. The marrow smears were cellular. Blasts showed high N:C ratio, moderate amount of cytoplasm, open chromatin and 2-3 nucleoli. Reaction, was normoblastic, megakaryopoiesis was severely diminished in the cases.

Multiple Myeloma: was established in one case of bone marrow aspirate. Bone marrow aspirate

showed infiltration by typical and atypical plasma cells.

Non Hodgkins lymphoma: bone marrow was hypercellular, with increased N: C ratio with blasts lymphoblast showing scant cytoplasm with indistinct nucleoli, with coarse chromatin with lymphocytosis.

The study titled” Clinico Haematological study in cases of pancytopenia” was carried out in the department of pathology of Jawaharlal Nehru Medical College, Sawangi (M) from July 2014 to august 2016. Total 53 cases were subjected for clinical and haematological study. Patients selected were those who were diagnosed as pancytopenia on peripheral blood smear and were subjected for bone marrow aspiration. Examination of the bone marrow is a key element in diagnosing many haematological and non-haematological disorders. Patients having pancytopenia due to the effect of chemotherapy, radiotherapy and immunosuppressant were excluded. Pancytopenia can result from a failure of production of hematopoietic progenitors, their destruction, or replacement of the bone marrow by tumor or fibrosis. Although selective cytopenias are important clinical entities, pancytopenia is a loss of all marrow elements Careful examination of peripheral blood smear for RBC, leukocyte and platelet morphology are important. Bone marrow examination is a simple and safe invasive procedure, which causes a moderate discomfort and can be performed easily. Its greatest utility is for investigating and it is an important diagnostic modality for evaluating the causes of pancytopenia.[10]. In the present study male to female ratio was 1:1.12 and there was a female preponderance (54.28%). Present study correlated well with the finding of Kumar and Raghupati et al. (2012)^[28], tariq ajij et al.(2010)^[29] they also found females to be commonly affected in their study.

Pancytopenia in Different Age Groups:-

In the present study highest number of patients i.e. 15 (28.30%) belong to the age group of 21-30 years, followed by 10 patients (18.87%) in the age group of 31-40 years. Present study has similar findings and it is in close proximity with the studies done by Tariq Ajij et al. (2010)^[29], Pathak Jha et al. (2012)^[27]

Table 6: Bone marrow diagnosis in various studies on pancytopenia

In the present study out of total 53 cases the commonest cause was found to be megaloblastic anaemia i.e 52.8%, followed by acute leukemia i.e 20.8% and normoblastic erythroid hyperplasia 11.3% aplastic anaemia was found to be 9.4%. The present study was in concordance with kirpal das et al.(2013) who also found the commonest cause was megaloblastic anaemia 41.9%, followed by acute leukemia27.4%, and aplastic anaemia 19.4%. (as shown in table 6)

In our study isolated megaloblastic anemia was the most common cause. Megaloblastic anemia is a group of disorder characterized by the presence of distinctive morphologic appearances of the developing red cell in the bone marrow. The commonest cause of megaloblastic anemia is folate and cobalamine deficiency and rarely by genetic or acquired abnormalities affecting the metabolism of the vitamin or because of defect in DNA synthesis not related to cobalamine or folate. Diagnosis of megaloblastosis in this study was established by bone marrow findings and further estimation of folic acid and vitamin B12 levels was not performed. The exact cause of deficiency of these vitamins was also not detected, although it was in sharp contrast with the study of Jha et al. (2008)[30] who found aplastic anemia to be the commonest cause this could be because the reported incidence of aplastic anemia varies considerably between countries e.g. from 0.7 to 4.1 per million per year in one study. Incidence is lower in Europe and North America than in various other parts of the world e.g. Asia.

Table 7: Compsarison according to physical findings in patients presenting with pancytopenia

In the present study, Pallor was seen in 100% followed by splenomegaly (56.60%) and hepatomegaly (35.85%,lymphadenopathy in 20.75%, petechial haemorrhage were present in 18.87%. The present study was in concordance with the findings of Kishor Khodke et al. (2001)^[31] and Soma Yadav et al. 2017^[34] (as shown in table 7)

Table 8: Cellularity of bone marrow in cases of pancytopenia:-

In the present study Out of 53 cases, Hypercellular marrow was observed in 42 cases (79.24%) followed by normocellular 6 (11.32%) and hypocellular marrow in 5 cases (9.43%) respectively. The present study is in concordance with the findings of Kalpana Chandra et al.^[36], and Rangaswamy et al.^[35] (as shown in table 8)

In conclusion, pancytopenia is a common entity. However, it has received an inadequate attention in the Indian subcontinent. A study of pancytopenia using easily available diagnostic techniques is therefore important for early diagnosis and timely management of patients. Megaloblastic anemia was the commonest cause of pancytopenia in the present study followed by acute leukemia, normoblastic erythroid hyperplasia and aplastic anemia among the non-malignant disorders. Acute leukemia constitutes the most common malignant haematological disorder causing pancytopenia. A comprehensive clinical, haematological workup and bone marrow study of patients usually help in evaluating the aetiology of pancytopenia. In addition attempt should be made for an early rec-ognition of underlying aetiology so that treatable causes are identified without delay and prognosis can be improved. Variation in the frequency of disorders causing pancytopenia has been ascribed to differences in methodology, stringency of diagnostic criteria, geographic area, period of observations, genetic differences, and varying exposure to cytotoxic agents after analysing the observations noted in the present study.

This study emphasized that in developing countries like India majority of the patients had reversible etiology and patients can be put on a trial of hematinics and close haematological follow up. Causes of pancytopenia varies widely according to various geographic regions. It can range from transient marrow suppression to malignancy. Bone marrow examination is an important tool in the diagnosis and early initiation of treatment in vulnerable pancytopenic patients. Bone marrow aspiration coupled with trephine biopsy can diagnose majority but not all the cases of pancytopenia. Hypoplastic anemia, hematological malignancies and megaloblastic anemia are the commonest causes of pancytopenia. Maximum diagnostic yield can be achieved by correlation with clinical findings, peripheral blood findings and with other laboratory and radiological parameters. Bone marrow aspiration is fairly safe procedure with minimal discomfort to the patient. Bone marrow aspiration is useful in the differential diagnosis of cytopenias. In leukaemia, bone marrow aspiation gives an assessment of marrow cellularity, presence of fibrosis.

In our country main cause of pancytopenia being fortunately megaloblastic anaemia which responds very well to treatment if diagnosed correctly in time. The present study concludes that detailed haematological investigations along with bone marrow examination in cytopenic patients are not only helpful in understanding the disease process but also to diagnose or to rule out the causes of pancytopenia and planning further investigations and management of these patients.

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