A clinico-hematological study in cases of pancytopenia: correlation of automated cell counter parameters in various etiologies.
Therefore bone marrow examinations such as bone marrow aspiration and biopsy are extremely helpful in evaluating the cause of pancytopenia in order to prevent grave complications and mortality as the underlying pathology determines the management and prognosis of the patients (4). This study therefore aims at establishing a correlation between clinical history, examination of peripheral blood finding, biochemical findings and bone marrow findings along with an analysis of automated cell counter parameters that can be of help in analyzing the possible causes of pancytopenia and thereby will automatically enhance the management process.
MATERIAL AND METHOD: This was a prospective study of one year duration from July 2011 to July 2012. Patients diagnosed as a case of pancytopenia having Hemoglobin less than 10 gm/dl, Total leucocyte countless than 4000/cumm, Platelets less than 100,000/cu. mm. were taken in the study.
STATISTICAL ANALYSIS: Continuous data were summarized as Mean [+ or -] SD while discrete (categorical) in %. The groups were compared by one way analysis of variance (ANOVA) followed by Tukey's post hoc test after ascertaining the normality by Shapiro-Wilk test and the homogeneity of variance by Levene's test. The categorical variables were compared by chi-square ([chi square]) test. Pearson correlation analysis was used to assess association between the variables. A two-sided ([alpha] = 2) p < 0.05 was considered statistically significant. All analyses were performed on STATISTICA (window version 6.0).
RESULTS: The present study was carried out in the Department of Pathology, in a medical institute, to study the underlying etiology in patients presenting with pancytopenia. A total of 60 cases presenting with pancytopenia were enrolled in the study.
Most of the patients belong to lower age ([less than or equal to] 30 yrs) groups (73.30%) with maximum number of patients were [less than or equal to] 10 yrs (30.0%) (figure 1). Male to female ratio was 1:1.2.
Most common etiology of pancytopenia was Aplastic anemia (38.3%), Megaloblastic anemia (21.7%) and Acute leukemia (13.3%), minor causes being Myelodysplastic syndrome, Hypersplenism, Non-Hodgkin's lymphoma, Leishmaniasis, Metastatic infiltration, HIV disease, Hemolytic anemia and Drug induced hypoplasia (figure 2).
The most common clinical manifestations were fever (100%), pallor (100%), and weight loss (100%) at the time of enrolment. Less common were neurological (3.2%), and bleeding from various sites as skin, nose, and gastrointestinal tract. Hepatomegaly (22.7%), splenomegaly (25%) and lymphadenopathy (11.7%) were also seen
Peripheral smear findings are discussed in table 1.
Diagnosis n Immature Hyperseg- Immature erythroid mented WBC precursor neutrophil Aplastic anemia 23 -- -- -- Megaloblastic anemia 13 3 5 -- Acute leukemia 8 -- -- 8 Hypersplenism 2 -- -- -- MDS 3 -- -- 1 Non-Hodgkins lymphoma 3 2 -- 1 Hepatitis 2 -- -- -- Leishmaniasis 2 -- -- -- Hemolytic anemia 1 -- -- -- HIV associate 1 -- -- -- Malignant infiltration 1 -- -- -- Drug induced 1 -- -- -- Total 60 Diagnosis Lympho- Increased Anisopoi- cytosis reticulo- kilocytosis cytes Aplastic anemia 21 -- 4 Megaloblastic anemia 9 2 9 Acute leukemia 7 1 4 Hypersplenism 2 -- 1 MDS 3 -- 1 Non-Hodgkins lymphoma 3 -- 1 Hepatitis 2 -- 1 Leishmaniasis 2 -- 2 Hemolytic anemia -- 1 1 HIV associate 1 -- 1 Malignant infiltration 1 -- 1 Drug induced 1 -- 1 Total
For further analysis, causes of pancytopenia have been grouped into: Aplastic anemia, Megaloblastic anemia, pancytopenia due to Infiltration (which comprises of acute leukemia, Non-Hodgkin's lymphoma, metastatic infiltration and MDS), pancytopenia due to other causes (all infectious causes- Leishmaniasis, hepatitis, hypersplenism, HIV reactive, and single case of hemolytic anemia and single case of drug induced pancytopenia).
Patients of aplastic anemia had significant lymphocytosis (p < 0.05) compared to megaloblastic (76.74 [+ or -] 15.06 vs 44.62 [+ or -] 20.44, p = 0.000), infiltration (59.07 [+ or -] 26.40, p = 0.043), other causes (44.56 [+ or -] 15.36 p = 0.001).
Very low Total Leucocyte Count (< 1000/cu.mm) was seen in Aplastic anemia and infiltration as ([chi square] = 13.51, p = 0.036) as compared to Megaloblastic anemia(1000-4000/cu.mm).
Lower Platelets counts (< 20,000/cu.mm) significantly (p < 0.001) associated with Aplastic anemia and Infiltration while higher Platelets counts (> 20,000/cu.mm) with the Megaloblastic anemia ([chi square] = 29.76, p < 0.001).
The normal (83-99 fl) values of MCV was significantly (p < 0.001) associated with Aplastic anemia and Infiltration while more than > 100fl especially with the Megaloblastic anemia ([chi square] = 25.39, p < 0.001) (table2)
However, no significant difference was found in MCH and MCHC values in different causes of pancytopenia.
The normal (11.6-14.0%) RDW-CV values were significantly (p < 0.001) associated with Aplastic anemia while higher (> 14%) with the Megaloblastic anemia and Infiltration ([chi square] = 18.93, p < 0.001) (table 3)
The normal values (6-13fl) of MPV is significantly (p < 0.001) associated with Aplastic anemia, Infiltration and Other causes of pancytopenia while higher (> 13%) with the Megaloblastic pancytopenia ([chi square] = 20.25, p < 0.001) (table 4)
Bone marrow cellularity is significantly associated with different causes of pancytopenia. Bone Marrow Hypocellularity showed a significant association with Aplastic anemia while both Normocellularity and Hypercellularity showed association with Megaloblastic anemia and Infiltration ([chi square] = 48.23, p < 0.001) (table 5)
Five cases were normocellular on aspiration, of which two cases (40%) turned out to be hypercellular and one (20%) hypocellular on trephine biopsy ([chi square] = 23.23, p = 0.001). Of the ten cases that were hypocellular on aspiration, one case showed normo-cellularity on biopsy along with focal bone marrow necrosis. Two cases that could not be aspirated due to dry tap were both hypercellular on biopsy. Thus concluding that bone marrow biopsy allows a better assessment of cellularity in addition to diagnosing the patient with dry tap on aspiration.
B12 and Folate deficiencies assessment were done in 11 out of 13 patients of megaloblastic anemia. Among, Megaloblastic anemia patients, the prevalence of B12 deficiencies was the highest (41.7%) followed by combined deficiency (25.0%) (Figure 3).
DISCUSSION: Our study group consisted of a total of 60 cases diagnosed as pancytopenia having hemoglobin less than 10 gm%, total leucocyte count less than 4000/cu.mm and a platelet count of less than 1,00,000/cumm. As depicted in figure no. 1, maximum numbers of cases were up to 10 years of age whereas the second most commonly affected age group was between 11-20 years (23.3%). Khunger et al. reported maximum number of cases in the third decade of life (4). Male: female ratio (1:1.2) in our study was almost equal. This was similar to Khunger et al. who reported a M:F ratio of 1.2:1. (4)
As shown in figure 2, on the basis of bone marrow examination the most common cause of pancytopenia in our study was Aplastic anemia (38.3% of patients). This is in concordance with the study of Varma et al., Kumar et al, Santra et al. (5, 6)
Whereas in studies of Khodke et al, Khunger et al, Tilak et al, Premkumar M et al, and Gayathri et al, megaloblastic anemia was the most important cause of Pancytopenia (2, 4, 7, 8) In another study by Pine et al., 64 children were identified with diagnosis of pancytopenia. The most common causes were infectious in origin (64%), followed by hematological (28%) and miscellaneous (8%) etiologies. (9)
The Pathophysiology of Aplastic anemia is believed to be immune mediated, with active destruction of blood forming cells by the lymphocytes. The aberrant immune response may be triggered by environmental exposures, such as to chemicals and drugs or viral infections and perhaps endogenous antigens generated by genetically altered bone marrow cells. This underlying mechanism is similar to other human disorders of lymphocyte mediated tissue specific organ destruction (diabetes, multiple sclerosis, colitis etc).
Acute leukemia was noted in 8 (13.4%) cases of pancytopenia in our study. Khunger et al. (4) reported an incidence of 5%. M. Premkumar et al. (9.2%) (7), BN Gayathri et al. (3.85%) (8), compared to Kumar R et al. (12%). (5) In our study among the eight leukemic patients, five were of ALL subtype. On flow cytometry, four of these leukemic patients were found to be of B cell type and there was a single case of T cell type leukemia. One case was of undifferentiated type. This patient expired within 4 days of the presentation and flow cytometric analysis was not done on the patient. The other two were cases of AML presenting in adults. On bone marrow aspiration of one of these we got a dry tap in one patient, she was subsequently subjected to biopsy which revealed AML probably arising from MDS, as there was evidence of dysplasia in the hematopoietic cells of the marrow along with the presence of myeloid blasts.
Zhou RH et al. in their article have stated that in case of AML, biopsy specimen will give more information than an aspiration smear about marrow cellularity, infiltration, presence of residual hemopoietic cells, qualitative and quantitative abnormalities of megakaryocytes showing myelodysplastic features. (10)
Thus concluding that bone marrow biopsy allows a better assessment of cellularity in addition to diagnosing the patient with dry tap on aspiration. The pathophysiology of pancytopenia in acute leukemia is unclear but is probably related to a combination of suppression of normal haematopoesis and replacement of bone marrow by leukemic cells resulting in pancytopenia and immunosuppression (11). Pancytopenia with few abnormal cells as seen in Myelodysplastic Syndrome was noted in 5% of our cases. In the study done by Khunger et al. (4) prevalence of MDS was found to be 2% similar to Kishor Khodke et al.(2%). (2)
MDS is characterized by peripheral pancytopenia despite a normocellular or hypercellular bone marrow because of increased apoptosis of hematopoietic bone marrow resulting in ineffective haematopoesis. Inhibition of apoptotic mechanism may induce leukemic transformation in MDS. (12) Pancytopenia related to Non Hodgkins Lymphoma was noted in 5% of the patients in our study as compared to the reported prevalence of 1% by Khunger et al. (4)
Pancytopenia in lymphoma was earlier thought to be due to two mechanisms: 1) Bone marrow infiltration 2) Hypersplenism. However, despite splenomegaly, hypersplenism has no major role in causing the cytopenias. The bone marrow is never sufficiently infiltrated to account for low peripheral blood counts. Evidence suggests that the neoplastic T cells cause suppression of haematopoesis through the lymphokines. Thus the main pathogenetic mechanism of cytopenias is the cell mediated suppression of normal haematopoesis. (13)
Among other causes of Pancytopenia, HIV was the important cause. Virtually all patients with advanced AIDS have pancytopenia as a rule the causes are production of the antibodies which might be triggered by the exposure of crypt antigens as a consequence of infection related damage of blood cells especially platelets and granulocytes. The haematopoietic cells especially platelets and granulocytes are antigenically similar to agents like HIV and other micro-organisms infecting the patients. These antibodies could interact with tissue antigens. Third possibility is that HIV act as the direct inducer of autoimmunity. (14, 15)
In our study 23% and 31% of the patients with Megaloblastic anemia presented with splenomegaly and hepatomegaly respectively along with two patients (15.3%) with lymphadenopathy. Osama Ishtiaq et al. in their study found 15.4% and 17.9% of megaloblastic anemia with splenomegaly and hepatomegaly respectively. (18)
As noted in our study as well as in study of Gayathri et al (8) Khunger et al (4) bone marrow lymphocytosis is more in aplastic anemia than Pancytopenia due to other causes.
A significant finding in our study was that the patients of aplastic anemia had a normal Mean Platelet Volume in 87% of the patients, as compared to megaloblastic anemia which shows higher than normal values in 70% of the patients as shown in table. Patients with acute leukemia and MDS had normal range. So MPV may be used as a parameter to differentiate megaloblastic pancytopenia from non-megaloblastic pancytopenia. MPV in Megaloblastic Anemia is significantly increased (p < 0.05) as compared to Aplastic Anemia (13.72 [+ or -] 1.75 vs 9.18 [+ or -] 2.83, p = 0.000), infiltration (10.07 [+ or -] 2.39, p = 0.001), and all other causes of pancytopenia (9.33 [+ or -] 1.21, p = 0.000) as shown in figure. This was in concordance with the study done by Chandra H et al (19).
Similarly Platelet distribution width (PDW) is significantly raised in patients with MDS as compared to all non myelodysplastic causes of pancytopenia.
In our study as well in study done by M Premkumar et al, the most important cause of megaloblastic Pancytopenia was cobalamine deficiency (7). Thus these studies reveal the multifactorial causation of Pancytopenia in our population is probable and occult cobalamine deficiency may contribute to the burden of hematological disease in patients with other primary diagnosis.
CONCLUSION: Pancytopenia is a common haematological problem encountered in clinical practice and should be suspected on clinical grounds when a patient presents with unexplained anaemia, prolonged fever and tendency to bleed. The present study concludes that detailed primary haematological investigations along with bone marrow aspiration, trephine biopsy and biochemical investigations are helpful for understanding the disease process to diagnose, or to rule out the causes of cytopenia, and in planning further investigations and management of cytopenic patients. Proper diagnostic work up is essential before use of hematinics and blood transfusion in all patients presenting as pancytopenia. Limitations of this study include lack of facilities and financial constraints.
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Soma Yadav , Rashmi Kushwaha , Kamal Aggrawal , A.K Tripathi , U.S Singh , Ashutosh Kumar 
[1.] Junior Resident, Department. Of pathology, King George's Medical University
[2.] Assistant Professor, Department. Of pathology, King George's Medical University
[3.] Professor, Department. Of pathology, King George's Medical University
[4.] Professor and Head, Department. Of Clinical Hematology, King George's Medical University
[5.] Professor, Department. Of pathology, King George's Medical University
[6.] Professor and officer in charge, Lymphoma--Leukemia Lab, Department. Of pathology, King George's Medical University.
Dr. Rashmi Kushwaha, King George's Medical University, Lucknow.
Table 2: Distribution of the MCV in different causes of pancytopenia Diagnosis No. of < 83fl 83-99fl cases Aplastic anemia 23 4 12 Megaloblastic anemia 13 0 0 Infiltration 15 5 7 Others 9 3 1 Total 60 12 26 Diagnosis > 100fl [chi square] p value value (DF = 6) Aplastic anemia 7 25.39 p < 0.001 Megaloblastic anemia 13 Infiltration 3 Others 5 Total 22 Table 3: Distribution of the RDW-CV in the different causes of pancytopenia Diagnosis No. 11.6-14% > 14% [chi square] p value of value cases (DF = 3) Aplastic 23 16 7 18.93 p < 0.001 anemia Megaloblastic 13 1 12 anemia Infiltration 15 2 13 Others 9 3 6 Total 60 22 38 Table 4: Distribution of the MPV in the different causes of pancytopenia Diagnosis No. 6-13fl > 13fl [chi square] p value of value cases (DF = 3) Aplastic 23 20 3 anemia Megaloblastic 13 4 9 20.25 p < 0.001 anemia Infiltration 15 13 2 Others 9 9 0 Total 60 46 14 Table 5: Cellularity in different causes of pancytopenia in the aspiration Diagnosis No. of Hypocellular Normocellular cases Aplastic 23 23 0 anemia Megaloblastic 13 0 6 anemia Infiltration 15 1 7 Others 9 3 3 Total 60 27 16 Diagnosis Hypercellular [chi square] p value value (DF = 6) Aplastic 0 48.23 p < 0.001 anemia Megaloblastic 7 anemia Infiltration 7 Others 3 Total 17 Table 6: Studies on Pancytopenia: Study Country No. of Most common cases cause International Israel & 319 AA (52.7%) agranulocytosis Europe and aplastic 1987 anemia study group (16) Keisu and Israel & 100 Neoplastic Ost (17) Europe disease, 1990 radiation (32%) Khodke et India 50 MA (44%) al. (2) 2000 Kumar et India 166 AA (29.51%) al. (5) 2001 Khunger et India MA (72%) al (4) 2002 M. Premkumar (7) India 140 MA (60.7%) 2008 Santra et al (6) India 111 AA (22%) 2010 Gayatri BN et India 104 MA (70.04%) al (8) 2011 Present study India 60 AA (38.3%) 2012 Study Second common Other causes cause International MDS (4.5%) AML, KZ, TB, Storage agranulocytosis disease, MM, Lymphoma and aplastic anemia study group (16) Keisu and AA (19%) MA, SLE, Drug induced, Ost (17) Falciparum Malaria, TB, CLL, MM, MDS, PNH, HIV Khodke et HA (14%) KZ, MM, HIV, MDS, AML, TB, al. (2) Drug induced cytopenia. Kumar et MA (22.3%) Aleukemic Leukemia, al. (5) Lymphoma, MDS Khunger et AA (14%) Aleukemic Leukemia, MDS, al (4) KZ, NHL, Malaria, TB, MM, Myelofibrosis M. Premkumar (7) Leukemia (9%), HIV, TB, KZ, Malaria, MDS, AA (8%) CML, Gauchers, Metastatic carcinoma Santra et al (6) Hypersplenism MA, SLE, Drug induced, (11%) Falciparum Malaria, TB, CLL, MM, MDS, PNH, HIV Gayatri BN et HA (18.26) Subleukemic Leukemia, al (8) Malaria, MM, Storage disorders. Present study Megaloblastic Leukemia, non-Hodgkins anemia (21.7%) lymphoma, infiltration, MDS Fig. 1: Age distribution of cases with pancytopenia. Ages (yrs) [less than or equal to] 10 Yrs 30.0% 11-20 Yrs 23.3% 21-30 Yrs 20.0% 31-40 Yrs 3.3% 51-60 Yrs 10.0% 61-70 Yrs 3.3% [greater than or equal to] 71 Yrs 1.7% Note: Table made from bar graph. Fig. 2: Etiological distribution of cases with pancytopenia. Etiological distribution of cases Aplastic anemia 38% Megaloblastic anemia 22% Acute leukemia 13% Myelodysplastic syndrome 5% Hypersplenism 3% Hepatitis 3% Non-hogkins lymphona 5% leishmaniasis 3% metastatic infiltration 2% HIV disease 2% Hemolytic anemia 2% Drug induced 2% Note: Table made from pie chart. Figure 3: Distribution of various deficiencies in megaloblastic anemia presenting with pancytopenia. B12 41.7% Folate 16.7% Combined 25.0% Normal B12 and Folate 16.7% Note: Table made from pie chart.
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|Title Annotation:||ORIGINAL ARTICLE|
|Author:||Yadav, Soma; Kushwaha, Rashmi; Aggrawal, Kamal; Tripathi, A.K.; Singh, U.S.; Kumar, Ashutosh|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Jun 3, 2013|
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