Bone Marrow Examination in Cases of Pancytopenia

One hundred and forty eight cases underwent bone marrow aspiration and it constituted 15.74% of total cases. Mean age was 30 years (range, 1-79 years). 42 cases were children (28.37%). Male: female ratio was 1.5:1. The commonest cause was hypoplastic bone marrow seen in 43 cases (29%) followed by megaloblastic anemia in 35 cases (23.64%), and hematological malignancy in 32 cases (21.62%). Erythroid hyperplasia was seen in 29 cases (19.6%) and normal bone marrow was seen in 5 cases (3.38%). There was one case each of Niemann-Pick disease and metastatic neuroblastoma in children and chronic pure red cell aplasia and leishmaniasis in adults. Acute leukemia was the commonest hematological malignancy. In children, commonest fi nding was hypoplastic bone marrow (38.1%) while in adults megaloblastic anemia (30.18%) was commonest fi nding followed by hypoplastic anemia (25.47%).


INTRODUCTION
Pancytopenia is defi ned by reduction of all the three formed elements of blood below the normal reference range. 1 The presenting symptoms are often attributable to the anemia or the thrombocytopenia.Leucopenia is often seen in the subsequent course of the disorder. 2arieties of hematopoietic and non-hematopoietic conditions manifest with features of pancytopenia.The underlying mechanisms are decrease in hematopoietic cell production, marrow replacement by abnormal cells, suppression of marrow growth and differentiation, ineffective hematopoiesis with cell death, defective cells formation which are removed from the circulation, antibody mediated sequestration or destruction of cells, and trapping of cells in a hypertrophied and overactive reticuloendothelial system. 1,3 cases of ineffective hematopoiesis the marrow may be normocellular or hypercellular. 1 Bone marrow examination is extremely helpful in evaluation of pancytopenia. 4Few similar studies have been published in the country. 5This study was carried out with an aim to obtain further information so that it would help in the management of patient with pancytopenia.

MATERIALS AND METHODS
The present study was a cross-sectional study, conducted at the Department of Pathology, Tribhuvan University Teaching Hospital (TUTH), Kathmandu, Nepal, over a period of 2 years (April 2005 to March 2007).The inclusion criteria for pancytopenia were hemoglobin (Hb) less than 10 gm/dL, total leukocyte count (TLC) less than 4000/cumm and platelet count less than 140000/cumm. 3,6Cases fulfi lling the defi nition of pancytopenia but lacking marrow representative bone marrow in the marrow aspirate were also excluded from the study.In each case bone marrow aspiration (BMA) was performed by the standard technique using Salah needle, from the posterior iliac crest under local anesthesia with standard aseptic precautions.Wright stain was used to stain all bone marrow smears.When required cytochemical stains such as Periodic-Acid Schiff (PAS) and Myeloperoxidase (MPO) stains were also used.The necessary hematological and clinical parameters were also noted.

RESULTS
The total number of bone marrow examination (BMEs) performed over two years for various indications at the Department of Pathology was 940.In 163 (17.34%) cases, bone marrow examination was performed for the indication of pancytopenia.Fifteen cases were excluded from the study as there was no representative marrow in the smears.Thus the total number of cases included in the study was 148 (15.74%).There were 89 males and 59 females with male: female ratio of 1.5:1.The mean age was 30 years with range of 1-79 years.Bone marrow aspiration fi ndings and distribution of the patients are shown in Table 1.In cases of hypoplastic bone marrow, 25 cases were male and 18 cases were female with male: female ratio of 1.3:1.The age ranged from 1.5-70 years (median 17 years).In cases of megaloblastic anemia, 21 were male and 14 were female with male: female ratio of 1.5:1.The age ranged from ten to seventy nine years (median 31 years).
Hematological malignancies are shown in Table 2, Acute leukemia alone constituted 90.62% of all hematological malignancies.Nine cases were acute lymphoblastic leukemia (ALL) and 20 cases were acute myeloid leukemia (AML).In cases of acute leukemia age ranged from 2-75 years with median of a 22 years.The male: female ratio was 1.9:1.The patient profi le and hematological parameters of ALL and AML are  3. AML-M2 was the most common type of AML (n=12) followed by AML-M3 (n=5), AML-M4 (n=2), AML-M5 and AML-M6 one each.There were fi ve cases of ALL-L1 and four cases of ALL-L2.
A comparison of age of the patients and hematological parameters of four major bone marrow fi ndings are show in Table 4.In the pediatric age group (≤14 years) hypoplastic anemia was the commonest fi ndings followed by erythroid hyperplasia, acute leukemia, while megaloblastic anemia was fourth most common (Table 5).In adults (15 years and above), megaloblastic anemia was the most common fi nding followed by hypoplastic marrow, hematological malignancy and erythroid hyperplasia.

DISCUSSION
Bone marrow examination for the evaluation of pancytopenia is a frequently requested investigation.In the present study, 17.34% of the total BMA cases was indicated for evaluation of pancytopenia.However, 15.74% cases were enrolled in this study.In a study conducted by Bashawri et al, the frequency of BME for evaluation of pancytopenia was 11.9%. 7Bone marrow In the present study, BME alone provided specifi c diagnosis in 77% of cases (Table 1 & 2).The commonest bone marrow fi nding was hypoplastic anemia followed by megaloblastic anemia.The frequency of various diagnostic entities causing pancytopenia has been attributed to differences in the methodology and stringency of diagnostic criteria, geographic differences, and underlying exposure to myelotoxic drugs. 8 In the present study the frequency of various diagnostic entities seen in the cases of pancytopenia in ≤15 years of age and ≥15 years of age is shown in Table 5.In a study conducted by Bhatnagar et al in children, megaloblastic anemia was the commonest cause of pancytopenia (28.4%) followed by acute leukemia (21%) and hypoplastic anemia (20%). 14Hematological parameters in the major causes of pancytopenia show overlap without any clue to diagnosis (Table 4).Similar results have been obtained by Kumar et al. 6 In the present study the diagnosis of hypoplastic anemia in all 43 cases was given based on BMA fi ndings and only in 22 was cases bone marrow biopsy available.It is recommended that both aspiration and trephine biopsy be done simultaneously in cases of pancytopenia. 6spiration smears are superior for morphological details while biopsy provides a more reliable index of cellularity and often reveals bone marrow infi ltration, fi brosis and granulomas. 6Occasionally, in cases of hypoplastic anemia marrow may be very cellular but the smears show depleted megakaryocytes, which is because of a regenerating focus called "Hot spot".In such a situation, bone marrow biopsy can be diagnostic. 1 Megaloblastic anemia was a common diagnosis in the present study as well as in other studies. 3,11,14In the present study, we could not identify the exact defi ciency leading to megaloblastic anemia as serum folic acid or vitamin B12 levels were not assessed.The higher numbers of megaloblastic anemia seem to refl ect higher a prevalence of nutritional defi ciency in our country.Acute megaloblastic anemia is an important cause of pancytopenia occurring rapidly in critically ill patients due to acute folic acid defi ciency. 6 the present study, hematological malignancy as a cause pancytopenia was found in signifi cant numbers.The most common condition was acute leukemia.In children, acute leukemia was the only hematological malignancy, while in the adult , acute leukemia constituted 20 out of 23.Nevertheless, in the study of Bashawri the main indication for BME in cases of pancytopenia was investigation of acute leukemia. 7Acute leukemia was the third common cause of pancytopenia in the study of Varma and Dash. 4 Similarly, in the study of Savage et al in Zimbabwe, acute leukemia constituted the third most common cause of pancytopenia.In their study, patients with hypoplastic anemia and acute leukemia were usually children, whereas those with megaloblastic anemia were adults, 12 similar to this study (Table 5).ALL was the commonest acute leukemia in the pediatric age while AML was the commonest in adults.Only three cases of ALL (two cases of ALL-L2 and one case of ALL-L1) were seen in dults.And this fi nding supports the well-established fi nding that ALL is common in children (Table 3).ALL-L1 was the most common type of ALL while AML-M2 was the most common type of AML, and most of them were seen in adults.In children, one case each of AML-M2, AML-M-3 and AML-M4 were seen.Hematological parameters of ALL and AML show considerable overlap (Table 3).
Erythroid hyperplasia was one of the common fi ndings in the present study and its relationship to pancytopenia is uncertain.Some of these cases may represent one phase in the evolution of hypoplasia, while some may be cases of refractory anemia.The criteria for differentiation of these groups remain unsatisfactory and these patients should be kept under follow-up. 3Hypercellular or normocellular marrow in cases of pancytopenia can also be seen in cases with ineffective hematopoiesis with cell death within the marrow. 1 Similarly, hypercellular marrow in the presence of peripheral pancytopenia can be a manifestation of paraneoplastic syndrome. 15ishmaniasis was seen in only one case, but it is not a rare fi nding in the studies from India.

Table 6 . A comparison of the fi rst and second most common causes of pancytopenia in different studies
Similarly, in the studies by Khodke et al and Tilak et al one case of AML was detected as a cause of pancytopenia. 3,

3,6,11 Metastatic
neuroblastoma and Niemann-Pick disease were rare causes of pancytopenia in the present study.Another rare but interesting fi nding in this study was chronic PRCA causing pancytopenia.Chronic PRCA usually manifests as anemia but rarely can have pancytopenia. 1CONCLUSION BME is an established diagnostic modality in the evaluation of pancytopenia.BME in most cases gives the specifi c diagnosis.However, in a few cases, additional tests are required.In the present study, BME was able to establish the diagnosis in 77% of cases.Hypoplastic marrow was the commonest diagnosis, followed by megaloblastic anemia, and hematological malignancies.However, in adults, megaloblastic anemia was the commonest fi nding followed by hypoplastic anemia.Rare causes included metastatic neuroblastoma, chronic PRCA, Niemann-Pick disease and leishmaniasis.Hypercellular marrow with erythroid hyperplasia and normal marrow were seen in 19.6% and 3.38% of cases respectively, which may require additional tests.