govindaraj t

BONE MARROW STUDY IN ADULTS WITH PANCYTOPENIA

by

Dr. GOVINDARAJ T.,M.B.B.S. Dissertation Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore In partial fulfillment

of the requirements for the degree of

DOCTOR OF MEDICINE in

PATHOLOGY

Under the guidance of

Dr. A.L. HEMALATHA,MD,DCP Professor and Head

DEPARTMENT OF PATHOLOGY MYSORE MEDICAL COLLEGE AND RESEARCH INSTITUTE

MYSORE-570 001

APRIL 2011

ii

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,

KARNATAKA, BANGALORE

DECLARATION BY THE CANDIDATE

I hereby declare that this dissertation entitled “BONE MARROW STUDY IN

ADULTS WITH PANCYTOPENIA” is a bonafide and genuine research work carried

out by me under the guidance of Dr. A.L. HEMALATHA,MD,DCP, Professor and Head,

Department of Pathology, Mysore Medical College and Research Institute, Mysore,

Karnataka.

iii

CERTIFICATE BY THE GUIDE

This is to certify that the dissertation entitled “BONE MARROW STUDY

IN ADULTS WITH PANCYTOPENIA” is a bonafide research work done by

Dr. GOVINDARAJ T., in partial fulfillment of the requirement for the degree of Doctor

of Medicine in Pathology.

iv

ENDORSEMENT BY THE HEAD OF THE DEPARTMENT AND THE PRINCIPAL

This is to certify that the dissertation entitled “BONE MARROW STUDY

IN ADULTS WITH PANCYTOPENIA” is a bonafide research work done by

Dr. GOVINDARAJ T., under the guidance of Dr. A.L. HEMALATHA,MD,DCP,

Professor and Head, Department of Pathology, Mysore Medical College and Research

Institute, Mysore.

v

COPYRIGHT

Declaration by the candidate

I hereby declare that the Rajiv Gandhi University of Health Sciences, Karnataka,

Bangalore shall have the rights to preserve, use and disseminate this dissertation in print

or electronic format for academic/research purpose.

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

vi

ACKNOWLEDGEMENTS

I express my deep sense of gratitude and indebtedness to my most respected guide

and teacher, Dr. A.L. Hemalatha, Professor and Head, Department of Pathology, Mysore

Medical College and Research Institute, Mysore as my guide and also for her constant

help, valuable advice, inspirational demeanor and able guidance throughout the study.

Her everwilling keenness to help and guide was a constant source of inspiration to me.

I heartily thank Dr. Bharathi M., Professor, Department of Pathology, Mysore

Medical College and Research Institute, Mysore for her help, valuable advice and

suggestions.

I owe my sincere thanks to Dr. Nataraju G., Professor, Department of Pathology,

Mysore Medical College and Research Institute, Mysore for his suggestions and advice.

My thanks to Dr. Sreenivas N., Dr. Ramesh B.H., Dr. Sharath Kumar,

Dr. Shilpa, Associate Professors, Department of Pathology, Mysore Medical College and

Research Institute, Mysore for their help and support.

My thanks to Dr. Umarani M.K., Dr. Shashidhar H.B., Dr. Adil S.A.K.,

Dr. Vani D., Dr. Gayathri M.N., Dr. Sumana, Dr. Vidya Vasudev, Assistant

Professors, Department of Pathology, Mysore Medical College and Research Institute,

Mysore, for their advice and suggestions.

I thank my wife Mrs. Kalaivani, and my senior Dr. Prabhu M.H., for their

constant help, encouragement and support.

I thank my other ever helping postgraduates and all technical and non-technical

staff of the department for the help extended.

vii

I also thank all the staff members, postgraduates of Medicine, Surgery

departments for their help and cooperation.

Above all, I thank the Almighty and all patients without whom this study would

not have been possible.

viii

LIST OF ABBREVIATIONS

AIDS Acquired Immuno Deficiency Syndrome

ALL Acute Lymphoid Leukemia

CFU Colony Forming Unit

DAF Decay Acceleration Factor

FAB French American British

GPI Glycosyl Phosphatidyl Inositol

HBsAg Hepatitis B Surface Antigen

IAHS Infection Associated Haemophagocytic Syndrome

M/E Myeloid Erythroid Ratio

MCV Mean Cell Volume

MDS Myelo Dysplastic Syndrome

MM Multiple Myeloma

NSAIDs Non-Steroidal Anti Inflammatory Drugs

PNH Paroxysmal Nocturnal Hemoglobinuria

RAEB-t Refractory Anemia with Excess Blasts in Transformation

RARS Refractory Anemia with Ringed Sideroblasts

SLE Systemic Lupus Erythematosus

TPO Thrombopoietin

TRAP Tartrate Resistant Acid Phosphatase

WBC White Blood Count

WHO World Health Organisation

ix

ABSTRACT BACKGROUND AND OBJECTIVE

The bone marrow, after the blood, is the largest and most widely distributed organ

in the body and is the principle site for blood cell formation. The spectrum of disorders

primarily or secondarily affecting the bone marrow may manifest with peripheral

pancytopenia. Hence bone marrow examination is extremely helpful in evaluation of

pancytopenia. This study highlights the spectrum of causes of pancytopenia and also

bone marrow morphology in cases of pancytopenia.

The objectives of the study are:

1. To study the spectrum of causes of pancytopenia in adults in the age group of

18-70 years.

2. To study the bone marrow morphology in cases of pancytopenia.

METHODOLOGY

Fifty patients with a hematological diagnosis of pancytopenia were studied during

the period November 2008 to May 2010, in the Department of Pathology, Mysore

Medical College and Research Institute, Mysore.

RESULTS

Out of 50 cases, 34 were males and 16 were females. Most of the patients were in

the age group of 41-60 years (54%). Megaloblastic anemia was the most important cause

of pancytopenia.

x

INTERPRETATION AND CONCLUSION

Bone marrow aspiration is relatively a very safe invasive procedure and

principally permits cytological assessment, since the underlying pathology determines the

management and prognosis of the patients.

KEY WORDS: Bone marrow aspiration; Pancytopenia

xi

TABLE OF CONTENTS

PAGE NO.

1. INTRODUCTION 1 2. OBJECTIVES 3 3. REVIEW OF LITERATURE 4 4. METHODOLOGY 39 5. RESULTS 49 6. DISCUSSION 74 7. CONCLUSION 85 8. SUMMARY 86 9. BIBLIOGRAPHY 89 10. ANNEXURES

(i) PROFORMA 98 (ii) STAINING TECHNIQUE 100 (iii) STATISTICAL METHODS APPLIED 102 (iv) KEY TO MASTER CHART 103

(v) MASTER CHART 104

xii

LIST OF TABLES Table No.

Title Page No.

1 Age and sex distribution of patients with pancytopenia 49

2 Clinical features 50

3 Range of hemoglobin in patients with pancytopenia 51

4 Range of leukocyte count in patients with pancytopenia 52

5 Range of platelet count in patients with pancytopenia 53

6 Range of reticulocyte count in patients with pancytopenia 54

7 Bone marrow cellularity in patients with pancytopenia 55

8 Age and sex distribution of patients with aplastic anemia 56

9 Range of hemoglobin in patients with aplastic anemia 56

10 Range of leukocyte count in patients with aplastic anemia 57

11 Range of platelet count in patients with aplastic anemia 57

12 Range of reticulocyte count in patients with aplastic anemia 58

13 Pancytopenia with hypercellular and normocellular marrow 59

14 Age and sex distribution of patients with megaloblastic anemia 60

15 Range of hemoglobin in patients with megaloblastic anemia 60

xiii

16 Range of leukocyte count in patients with megaloblastic anemia 61

17 Range of platelet count in patients with megaloblastic anemia 61

18 Range of reticulocyte count in patients with megaloblastic anemia 62

19 Age and sex distribution of patients in nutritional anemia 63

20 Range of hemoglobin in patients with nutritional anemia 63

21 Range of leukocyte count in patients with nutritional anemia 64

22 Range of platelet count in patients with nutritional anemia 64

23 Range of reticulocyte count in patients with nutritional anemia 65

24 Age and sex distribution in patients with hypersplenism 66

25 Range of hemoglobin in patients with hypersplenism 66

26 Range of leukocyte count in patients with hypersplenism 67

27 Range of platelet count in patients with hypersplenism 67

28 Range of reticulocyte count in patients with hypersplenism 68

29 Age and sex distribution of patients with leukemia 69

30 Range of hemoglobin in patients with leukemia 70

31 Range of total leukocyte count in patients with leukemia 70

32 Range of platelet count in patients with leukemia 71

33 Range of reticulocyte count in patients with leukemia 71

34 Causes of pancytopenia in various studies 76

xiv

LIST OF FIGURES Figure

No. Title Page

No.

1 Materials used for Bone Marrow aspiration and Biopsy 41

2 Bone Marrow aspirate particles on a glass slide 41

3 Normal bone marrow. (Leishman’s stain 10x X 10x) 42

4 Bone marrow aspiration smear showing Erythroid Hyperplasia (Leishman’s stain 10x X 10x ) 42

5 Megaloblastic anemia. Peripheral smear showing hypersegmented neutrophils and macroovalocytes. Leishman’s stain – 10x X 100x 43

6 Megaloblastic anemia. Bone marrow aspiration smear showing hypercellular marrow. Leishman’s stain – 10x X 10x 43

7 Megaloblastic anemia. Bone marrow aspiration smear showing megaloblasts with open chromatin. Leishman’s stain – 10x X 100x 44

8 Megaloblastic anemia. Bone marrow aspiration smear showing giant band form, mitosis and metamyelocyte. Leishman’s stain- 10x X 100x 44

9 Aplastic anemia. Bone marrow aspiration smear showing increase in fat cells. Leishman’s stain- 10x X 10x 45

10 Aplastic anemia. Bone marrow aspiration smear showing increase in fat cells. Leishman’s stain- 10x X 40x 45

11 Aplastic anemia. Bone marrow trephine biopsy showing increase in fat cells. H & E Stain- 10x X 40x 46

12 AML. Bone marrow aspiration smear showing myeloblasts. Leishman’s stain- 10x X 40x 46

xv

13 AML. Bone marrow aspiration smear showing myeloblasts positive for MPO.-10x X 100x 47

14 MDS. Bone marrow aspiration smear showing dyserythropoiesis and blasts. Leishman’s stain- 10x X 100x 47

15 MDS. Bone marrow aspiration smear showing dyserythropoiesis and mitosis. Leishman’s stain- 10x X 100x 48

16 Age and sex distribution of patients with pancytopenia 50

17 Range of hemoglobin in patients with pancytopenia 51

18 Range of leukocyte count in patients with pancytopenia 52

19 Range of platelet count in patients with pancytopenia 53

20 Range of reticulocyte count in patients with pancytopenia 54

1

INTRODUCTION

The bone marrow is the largest and most widely distributed organ in the body.

It is the principle site for blood cell formation. In the normal adult, its daily

production and export of blood cells amounts to about 2.5 billion red cells, 2.5 billion

platelets and 1.0 billion granulocytes per kilogram of body weight.1

The spectrum of disorders primarily or secondarily affecting the bone marrow

may manifest with peripheral pancytopenia.2 Pancytopenia is defined as reduction of

all the three formed elements of blood below the normal reference range. The

presenting symptoms are often attributable either to the anaemia or

thrombocytopenia. Leucopenia is often seen in the subsequent course of the disorder.

Varieties of hematopoietic and non-hematopoietic conditions manifest with features

of pancytopenia.

The mechanisms contributing to pancytopenia include, decrease in

haematopoietic cell production, marrow replacement by abnormal cells, suppression

of marrow growth and differentiation, ineffective hematopoiesis with cell death,

defective cell formation, antibody mediated sequestration or destruction of cells in a

hypertrophied and overactive reticuloendothelial system.3

Pancytopenia is a serious hematological problem, the underlying cause of

which is diagnosed by bone marrow aspiration and biopsy. Bone marrow examination

is extremely helpful in the evaluation of pancytopenia.4

2

Various factors encompassing geographic distribution and genetic

disturbances may cause variation in the incidence of disorders causing

pancytopenia.5,6,7 A few similar studies are available in literature.

Although it is a common clinical pattern with an extensive differential

diagnosis, there is little discussion of this abnormality in major textbooks of internal

medicine and haematology. Since the underlying pathology of pancytopenia

determines the management and prognosis of patients, there is a definite need to study

about pancytopenia.8

Cell composition of aspirated material from a normal adult bone marrow9

Erythroid series

Pronormoblast 0.1-1.1

Basophilic normoblast 0.4-2.4

Polychromatophilic normoblast 2-30

Orthochromatophilic normoblast 2-10

Granulocytic series

Myeloblast 0.1-3.5

Promyelocyte 0.5-5

Myelocyte 5-23

Metamyelocyte 7-27

Band form 9-18

Mature Neutrophils 4-28

Others

Lymphocyte 5-24

Plasma cells 0-3.5

Monocytes 0-0.6

Macrophages 0-2

Megakaryocyte 0-0.5

3

OBJECTIVES

1. To study the spectrum of causes of pancytopenia in adults in the age group of

18-70 years.

2. To study the bone marrow morphology in cases of pancytopenia.

4

REVIEW OF LITERATURE

Hematopoiesis involves the processes in the production of all the cells of

blood from hematopoietic cells. These processes include the self-renewal of stem

cells, the commitment of most progeny of stem cells to differentiate ultimately into a

particular cell type and the proliferation of progenitor cells and their differentiation to

a particular kind of mature blood cell.

MEGALOBLASTIC ANEMIA

A study was conducted in the department of hematology and transfusion

medicine, Government Medical College and Hospital, Chandigarh for over a period

of 32 months. During this period, pancytopenia was an indication for bone marrow

(aspiration and trephine) in 77 out of a total of 205 cases (37.6%). The most common

cause of pancytopenia as revealed by bone marrow examination was megaloblastic

anaemia (68%).10 Weston CF et al. (1987) reported pancytopenia and folate

deficiency in three alcoholics. Folate deficiency is a common finding in alcoholics

due to abnormalities in diet, intestinal absorption, internal metabolism and

excretion.11 Eighty patients with vitamin deficiency induced pancytopenia were

admitted within a year, and these vitamin deficiencies are frequent in underdeveloped

countries and are responsible for megaloblastic anaemia.12 Rizwan Aizz Qazi, Ayesha

Masood conducted a study on 100 consecutive patients with laboratory evidence of

pancytopenia, collected systematically over a period of 14 months beginning from

December 1999. The final diagnosis was based on bone marrow examination

findings. The most common cause was megaloblastic anemia (28%).13

5

The department of pathology, Dr.RML Hospital, New Delhi conducted a study

over a period of six months. During this period, a total of 250 bone marrow smears

were examined. Out of these, 50 aspirations were done in cases of pancytopenia with

an age range between 3-69 years and a male to female ratio of 1.3:1 (29/21). The

criteria for diagnosis of pancytopenia were,

1. Hemoglobin < 10 gm/dl

2. Total leucocyte count < 3500/cumm

3. Platelet count < 100000/cumm

In this study, the commonest cause of pancytopenia was megaloblastic

anaemia which was seen in 22 out of 50 patients (44%).2

In another study which was conducted over a period of one year in the

Department of Haematology, Safdarjung Hospital Medical College, New Delhi, to

ascertain the percentage of occurrence and causes of pancytopenia, bone marrow

aspirations / biopsies were performed in most of the cases (200 out of 250 cases). The

commonest cause of pancytopenia was Megaloblastic anaemia (72%).14

APLASTIC ANEMIA16

Haematopoiesis, a fundamental concept in haematology and Aplastic anaemia,

a disease due to the absence of haematopoiesis, has had parallel histories since the

discovery of the function of bone marrow in the mid 19th century.

The disease was named by Vaquez and Aubertin in 1904 as “Pernicious

anemia with yellow marrow”. They emphasized its pathophysiology of failed

hematopoiesis which they called “anhematopoiesis”. Neelmann and Bizzozero (1868)

6

observed nucleated erythroid cells in the marrow and concluded that it was the site of

continuously proliferating blood cells.

Paul Ehrlich (1888) correlated the absence of formed elements in the blood in

pregnant women, to severe marrow hypoplasia at autopsy. Cabot stressed the

marrow’s distinctive pathology and the need for its examination in the diagnosis.

Santesson (1897) recognised toxic substances such as Benzol as a cause of aplastic

anemia.

Smith (1919) reported pancytopenia as a well-defined clinical entity associated

with aplastic anemia. Rhoades and Miller (1938) showed that the marrow in cases of

aplastic anaemia varied in cellularity from aplasia to hypoplasia. Daniel et al.15

(1958), in their analysis of fifty cases of aplastic anaemia, reported forty-three cases

of idiopathic aplastic anaemia. The remaining seven cases were attributed to Benzol,

phenylbutazone, chloramphenicol and arsenic fruit spray.

Organic Arsenicals, gold compounds and radioactive compounds were

reported to cause aplastic anaemia. Numerous substances added to the list include

sulphonamides (Meyer, Perlmutter, 1942), Mepacrine (Custer, 1946; Parmer, 1948),

Streptomycin (Corelli, 1947) and Tridone.16 Adams EB (1951) reported pancytopenia

associated with idiopathic aplastic anaemia in twenty-seven cases. He also reported

pancytopenia with aleukemic leukaemia in three patients.17

Retief HP, Haynes AD (1971-1975)18 reported on 195 patients with

pancytopenia. Classic aplastic anaemia was found in 22 patients, with no apparent

etiology in 16, previous phenylbutazone ingestion in 2 and Fanconi’s anaemia in 4

patients. Aplastic anaemia associated with various drugs have been described which

7

include OKT3, ibuprofen and Ciprofloxacin.19 International Agranulocytosis and

Aplastic Anemia study (1986) confirmed the risk of Aplastic anemia with

phenylbutazone use and identified even higher probabilities with other NSAIDs.16

Puedssi et al. (1977)20 reported four cases of aplastic anaemia associated with

sub massive hepatic necrosis. These patients were HBsAg negative and the exact

cause could not be determined. Dennis et al. (1978)21 reported the association of

aplastic anaemia with type B viral hepatitis. These patients were positive for HBsAg.

Non A Non B hepatitis associated with aplastic anaemia was reported by Jerome et al.

(1979). Rafel M. et al. (1998)22 found transient pancytopenia after Non A, Non B and

Non C acute hepatitis preceding ALL.

Lorenz et al. (1955) from Australia first described association of aplastic

anaemia with viral hepatitis. Bierman HR and Nelson ER (1965) have reported

Dengue like viral infections in association with hypoplastic marrow.23 Pancytopenia

associated with infectious mononucleosis was reported by Kenneth et al. (1981),24

which was due to suppression of haematopoiesis by activated T-cells.

Young N, Mortimer P (1984)25 reported Parvovirus associated with aplastic

anaemia. Osaki et al. (1999)26 have reported severe aplastic anaemia with human

parvovirus B19 infection in a patient without any underlying disorder. Marrow

hypocellularity has been a common finding while aplastic anaemia has been described

rarely.

Aplastic anaemia is strongly associated with rare collagen vascular syndrome

called eosinophilic fasciitis. Pancytopenia with marrow hypoplasia can also occur in

Systemic Lupus Erythematosus (SLE). This may be due to folate deficiency

8

secondary to hemolysis, infections, treatment with drugs and autoimmunity. Pereira

et al.27 (1998) have noticed global hypocellularity (47.6%), increased reticulin

production (76.2%) and necrosis (19%) in 21 bone marrow specimens from patients

with SLE. They concluded that bone marrow might be a target organ in SLE with

cytopenias.

Paroxysmal nocturnal hemoglobinuria is a clonal disorder arising from a

somatic mutation in the haematopoietic stem cell. Erythrocytes, white cells and

platelets are affected by mutation, which renders their membranes highly susceptible

to lysis by complement. This abnormal sensitivity is predominantly due to a

deficiency in complement regulatory membrane proteins such as Decay Accelerating

Factor (DAF) and CD59 which are covalently attached to the cell membrane by a

glycosyl phosphatidyl inositol anchor (GPI). But the molecular mechanism of the

abnormal hemolysis is now being rapidly clarified, and the lack of GPI-anchored

membrane proteins has been shown to have diagnostic value in PNH.28

Two other syndromes associated with aplastic anaemia are, Dyskeratosis

congenita characterised by aplastic anaemia, reticulated hyperpigmentation, nail

dystrophy and mucosal leukoplakia described by Steier N et al.29 (1972) and

Schwachman-Diamond syndrome associated with pancreatic insufficiency,

pancytopenia and hypoplastic marrow described by Schwachmann H et al. (1964).

Fatal pancytopenia in falciparum malaria was reported by Arya TV, Prasad

RN,30 Yamakawa H et al.31 (1989) have reported a case of plasmodium vivax malaria

complicated with pancytopenia due to hypoplasia of the bone marrow. Plasmodium

9

vivax causing pancytopenia after allogenic blood stem transplantation in a patient

with chronic myeloid leukaemia was reported by Raina V.et al.32 (1998).

Patients with infection associated haemophagocytic syndrome (IAHS) have

fever, severe constitutional symptoms and blood cytopenias (usually pancytopenia). A

viral etiology has been demonstrated in many cases but other infections may

occasionally cause similar changes. Pancytopenia due to haemophagocytic syndrome

as the presenting manifestation of tuberculosis was described by Basu S et al.33

(2000). In 1993, Cassim KM et al. found pancytopenia associated with disseminated

tuberculosis, reactive histiocytic haemophagocytic syndrome and tuberculous

hypersplenism. Haemophagocytic syndrome presenting with pancytopenia as a

complication of visceral leishmaniasis was described by Gagnaire M et al.34 (2000), in

Typhoid fever by Udden MM et al.35 (1986) and Sood R et al.36 (1997).

MYELODYSPLASTIC SYNDROME37

Myelodysplastic syndromes (MDS) are a heterogeneous group of acquired,

clonal stem cell disorders characterised by:

• Occurrence mainly in elderly individuals

• Dysplasia of one or more haematopoietic cell lines with resultant characteristic

morphological abnormalities

• Ineffective erythropoiesis due to increased apoptosis, causing cytopenia of one or

more cell lines in peripheral blood, and

• Increased risk of transformation to acute myeloid leukaemia

• MDS was previously called as dysmyelopoietic syndrome, preleukaemic

syndrome, smoldering acute leukaemia, and oligoblastic leukaemia.

10

Juneja SK et al.38 (1983) studied haematological features of 118 cases of

primary myelodysplastic syndrome, at initial presentation. The utility and prognostic

significance of FAB classification was studied in 237 patients with myelodysplastic

syndromes by Kerkhofs H. et al.39 (1986). The most important prognostic factor was

the number of blast cells in blood and bone marrow. Seventeen cases of

Myelodysplastic syndrome (ten were primary and seven secondary to previous

radiotherapy and chemotherapy), characterised by severe bone marrow fibrosis and a

high number of megakaryocytes were studied by Giorgio et al.40 (1991). All the

patients had pancytopenia and they concluded that MDS with fibrosis might represent

a clinicopathological entity, which needs to be distinguished from other MDS

subtypes as well as from idiopathic myelofibrosis. An international scoring system for

evaluating prognosis in MDS was performed using cytogenetical, morphological, and

clinical data by Greenberg P et al.41 (1997). Thirty one patients with Myelodysplastic

syndrome were studied by Kini J et al.42 (2001) and they found Refractory Anaemia

with excess blasts in transformation (RAEB-t) to be the commonest subtype (29%)

and Refractory anemia with ringed sideroblasts (RARS) the least common (6.4%).

Although myelodysplastic syndromes occur predominantly in the elderly, paediatric

age group is also affected by MDS.

Delacretaz F et al.43 (1987) in their study of twenty eight patients of MDS, to

evaluate whether the morphologic criteria proposed by FAB group for marrow smears

could be applied to trephine biopsies, found that, the five MDS types proposed by the

FAB group could be reliably distinguished on bone marrow biopsy, with the

knowledge of peripheral blood blast and monocyte counts especially when the

11

aspiration was inadequate and biopsy ruled out aplasia or tumor infiltration as the

possible alternative causes of cytopenias. Presently, the WHO Classification-1999 is

followed.

• Refractory Anemia

o With ringed sideroblasts

o Without ringed sideroblasts

• Refractory cytopenia with multilineage dysplasia

• Refractory anaemia with excess blasts,

• 5q syndrome

• Myelodysplastic syndrome, unclassifiable.

HYPERSPLENISM44

Banti (1800) and Gristel (1866) explained the idea that spleen may produce ill

effects through exaggeration of its normal activities. Chauffard (1907) introduced the

term hypersplenism to refer to this concept. Pancytopenia is one of the well-known

hematologic manifestations of hypersplenism.

Green D et al.45 (1971) presented a case of Gaucher’s disease with hypersplenism

and pancytopenia. Johnson HA et al.46 (1989) in a review of 391 splenectomies

performed over a sixteen-year period, observed that pancytopenia and haemolytic

complications of the disease processes were the commonest indications for

splenectomy.

12

Cruck and Riser (1949) have suggested the following criteria for the diagnosis

of hypersplenism.

1. A massively enlarged spleen.

2. Depleted cell values including anemia, neutropenia and thrombocytopenia,

occurring either singly or in combination.

3. Bone marrow hyperplasia with regard to the cells, which are deficient in the

blood.

4. Demonstration of increase in cell values after splenectomy.

MALIGNANCY

Pancytopenia is present at initial diagnosis in acute megakaryoblastic

leukaemia. This is due to fibrosis, believed to be secondary to the increase in

megakaryocytes that secrete platelet mitogenic factor, which stimulates the

fibroblasts. Pancytopenia can also be seen in acute erythroleukaemia.47 This

syndrome of ‘preleukaemic aplasia’ appears to be a specific feature of common ALL.

Howel RB et al.48 (1982) reported a subset of patients of acute myelogenous

leukaemia presenting with pancytopenia and predominant granulocytopenia. Sultan C

et al.49 (1981) described eight patients with acute myelodysplasia and myelofibrosis.

Four cases were secondary to long term therapy with cytotoxic agents and four were

idiopathic. All cases presented with an abrupt onset of illness, absence of

organomegaly and severe pancytopenia. Bone marrow showed myelodysplastic

features and fibrosis.

Bloomfield CD et al.50 (1976) reported hematological parameters of Non-

Hodgkin’s lymphoma in hundred and forty adults. Marrow involvement in poorly

13

differentiated lymphocytic lymphoma was associated with pancytopenia in 93% of his

patients. Pancytopenia was found in 95% of patients with nodular lymphomas in

whom marrow involvement was present. Brenner B et al.51 (1985) reported severe

pancytopenia associated with angioimmunoblastic lymphadenopathy due to marked

marrow fibrosis. Takai K, Sanada M52 (2000) reported Angioimmunoblastic T cell

lymphoma associated with haemophagocytic syndrome.

Zidar BL et al.53 (1977) reported seven cases of Hairy cell leukaemia, a

chronic lymphoproliferative disorder presenting with splenomegaly, pancytopenia and

recurrent infection. Harvey M et al.54 (1978) in their clinical review of seventy-one

cases of hairy cell leukaemia found an association with pancytopenia in all the cases.

Chudgar U et al.55 (1991) studied nine patients of hairy cell leukaemia in five years

and found that the male to female ratio was 8:1. Weakness and fatigue (66%) was the

commonest presenting symptom and splenomegaly (66%) was the commonest

physical finding. Varying degrees of pancytopenia was a consistent feature.

Contreras et al.56 (1971) in their study of 4000 bone marrow aspirates and

biopsies, observed that bone marrow biopsy is superior to aspiration in the diagnosis

of metastatic carcinoma.

Wade and Stevenson57 first described bone marrow necrosis (BMN, in 1942,

in a patient with sickle cell disease who died of cerebral infarction. It was not until the

early 1970s that BMN was frequently diagnosed before death. This was due to the

rare character of the disease, the underawareness by the medical community of its

existence, and its sparse reporting in haematological literature.

14

Bone marrow necrosis is best defined as necrosis of myeloid tissue and

medullary stroma in large areas of haematopoietic bone marrow. This contrasts with

the findings of aplastic anaemia in which there is loss of myeloid tissue but no

destruction of reticular architecture. Failure of the microcirculation must be

considered as a critical event in these cases. In 1976, Kiraly and Wheby58 found

pancytopenia in thirteen patients with BMN on a total of 664 subsequent bone

marrow aspirations and/or biopsies (2 %) taken over a twelve-year period.

Necrosis of the bone marrow is a very rare condition with a prevalence

ranging from 0.15-7%. In a study of four paediatric patients with bone marrow

necrosis, all the four presented with pancytopenia. Pancytopenia can occur in acute

leukaemia, lymphomas affecting bone marrow, metastatic tumors, sickle cell disease,

tuberculosis, severe bacterial infections and shock.

15

ETIOLOGY OF PANCYTOPENIA16

I. Pancytopenia with hypocellular Bone marrow

• Acquired aplastic anaemia

• Inherited aplastic anaemia (Fanconi’s anaemia and others)

• Some myelodysplasia syndromes

• Rare aleukemic leukaemia (acute myelogenous leukaemia)

• Some acute lymphoblastic leukaemias.

• Some Lymphomas of bone marrow.

II. Pancytopenia with cellular Bone marrow

A. Primary bone marrow diseases

• Myelodysplasia syndromes

• Paroxysmal Nocturnal Haemoglobinuria

• Myelofibrosis

• Some aleukemic leukaemias

• Myelophthisis

• Bone marrow lymphoma

• Hairy cell leukaemia.

B. Secondary to systemic diseases

• Systemic lupus Erythematosus, Sjogren’s syndrome

• Hypersplenism

• B12 and folate deficiency (familial defect)

• Over whelming infection

16

• Alcohol

• Brucellosis

• Ehrlichiosis

• Sarcoidosis

• Tuberculosis and atypical mycobacteria

III. Hypocellular Bone marrow ± Cytopenia

• Q fever

• Legionnaire’s disease

• Mycobacteria

• Tuberculosis

• Anorexia nervosa, starvation

• Hypothyroidism

Classification of Aplastic anaemia

I. Acquired

A) Idipathic

B) Secondary

a) Irradiation

b) Drugs and Chemicals

- Regular effects

Cytotoxic agents/ benzene

- Idiosyncratic reactions

Chloramphenicol/NSAIDs/Antiepileptics/gold/other drugs and

chemicals

17

c) Viruses

- Epstein Barr Virus

- Hepatitis (Non A, Non B, Non C hepatitis)

- Parvovirus B19

- HIV (AIDS)

d) Immune disorders

- Eosinophilic fasciitis

- Hypoimmunoglobulinemia

- Thymoma / Thymic carcinoma

- Graft versus host disease in immunodeficiency

e) Paroxysmal nocturnal haemoglobinuria

f) Pregnancy

II. Inherited

• Fanconi’s anaemia

• Dyskeratosis congenita

• Schwachman-Diamond Syndrome

• Reticular dysgenesis

• Amegakaryocytic thrombocytopenia

• Familial aplastic anaemia

• Preleukaemia (Monosomy 7)

• Non-hematologic syndromes

(Down’s, Dubowitz, Seckel)

18

Causes of Hypersplenism59

• Portal hypertension with congestive splenomegaly

• Lymphomas

• Sarcoidosis

• Felty’s syndrome

• Lipid storage disease – Gaucher’s disease

• Kala-azar, chronic Malaria, ‘tropical splenomegaly’

• Bacterial infections – Tuberculosis, brucellosis

• Thalassaemia

• Chronic lymphocytic leukaemia

• Myelofibrosis

• Hairy cell leukaemia

Pathophysiology of Pancytopenia16

Blood cell production is an enormous and complex process in which a

hierarchial developmental progression of primitive, multipotential Haematopoietic

stem cells gradually lose one or more developmental potentials and ultimately

become committed to a single cell lineage, which matures into the corresponding

blood cell type. Haematopoietic stem cells are characterised by the ability to self

renew and differentiate into all mature blood lineages. They are also capable of

rescuing lethally irradiated hosts by reconstituting the entire repertoire of

hematopoietic cells in the recipients. Haematopoietic stem cells reside predominantly

within the bone marrow as quiescent, inactive blood cells, in contact with non

hematopoietic cells that make up the bone marrow microenvironment.

19

The passage of Haematopoietic stem cells through different stages of stem and

progenitor compartments requires the presence of soluble factors known as cytokines.

1. Regulation of Erythropoiesis

Erythropoiesis, in postnatal life, takes place within the environment of the

bone marrow which is mainly concentrated in the axial skeleton, ribs and pelvis.

In the steady state, the hematopoietic microenvironment is probably the most

important controlling aspect of erythropoiesis, with local cytokine release from the

bone marrow stroma and the binding of cytokines to the stromal matrix which

determines the rate of proliferation and differentiation. Erythropoietin, which is

produced predominantly by the kidneys in adults, stimulates the conversion of

Erythropoietin Responsive Cells (ERC) to pronormoblasts. Erythroid differentiation,

at least in the later stages, seems to have a relationship with macrophages, the so-

called ‘nurse cells’. Within the haematopoietic sinusoids, the maturing precursor cells

move towards the adventitial cells, which line the capillaries. Reticulocytes only

escape into the circulation when they are sufficiently deformable to move between the

adventitial cells.

The erythroid progenitor cells can be identified by the characteristics of the

erythroid colonies, which generate in appropriate semi-solid media. The earliest

recognisable committed progenitor for erythroid cells is the CFU-GEMM (Colony

Forming Unit-Granulocytes Erythroid, Megakaryocyte and Macrophage). The next

identifiable progenitor cells are the Burst Forming Units-Erythroid (BFU-E). This

BFU-E is divided into early and late compartments, depending upon the time taken to

establish the bursts in vitro. Significant numbers of BFU-E are normally present in the

20

peripheral blood. The final progenitor cell is identified as CFU-E. The Epo-R

(erythropoietin receptor) is expressed on the surfaces of all the committed erythroid

cells in the progenitor compartment with relatively low numbers on CFU-GEMM and

early BFU-E, increasing numbers in the intermediate and late BFU-E and maximal

expression in CFU-E. The binding of erythropoietin to Epo-R prevents apoptosis in

the CFU-E pool and the cell cycle progresses into Erythron which is the

morphologically recognisable erythroid cell compartment within the bone marrow

consisting of pronormoblasts, early normoblasts, intermediate normoblasts, late

normoblasts and finally, the extrusion of the nucleus, giving rise to the final stage-

reticulocyte.

2. Regulation of granulopoiesis and monopoiesis47

Granulocytes and Monocytes are derived from a common bipotential stem cell

(CFU-GM) which is derived from the CFU-GEMM.

1. CFU-GEMM IL-3 GM-CSF CFU-GM (Colony forming unit Granulocyte macrophage).

2. CFU-GM IL-3, GM-CSF CFU-G (Granulocytes) G-CSF

3. CFU-GM IL-3, GM-CSM CFU-M (Monocytes/Macrophages)M-CSF

4. CFU-GEMM CFU-Eo IL-3, GM-CSF , (Eosinophils) IL-5

5. CFU-GEMM CFU-Ba IL-3 (Basophils)

• GM-CSF-Colony Stimulating Factor – Granulocyte, Monocyte / Macrophage.

The available data suggests that, macrophages play a key role in regulating the

production of their own precursors as well as granulocytes by producing both Colony

Stimulating Factor (CSF) as well as Prostaglandin E (PGE), an inhibitor of CFU-E.

21

Granulocytes contain some inhibitory substances which may participate in the

regulation of production of monocytes and granulocytes in-vitro by providing a

negative feedback between the mass of mature granulocytes and the rate of

production of new cells.

3. Regulation of Megakaryopoiesis47

Megakaryocytes are derived from pleuripotent stem cells, the earliest

recognised platelet precursors being the burst forming units denoted by BFU-Meg.

Under the influence of thrombopoietin (TPO) and cytokines such as IL-3, and IL-11,

the BFU-Megs develop into megakaryocyte colony forming units (CFU-Meg). As the

CFU-Megs mature, they develop the morphological and biochemical features of

megakaryoblasts and further megakaryocytes. The fully mature megakaryocyte has

proliferation of the characteristic platelet granules (alpha-granules and dense bodies)

and membrane glycoproteins which are vital for platelet function. Thrombopoietin is

able to induce the complete sequence of maturation without the addition of any other

factors and may be considered as the master growth factor for thrombopoiesis, just as

erythropoietin is for the erythron.

4. Regulation of Lymphopoiesis47

The lymphoid stem cell is derived from pleuripotential stem cell and gives rise

to T and B lymphocytes which are morphologically identical, but immunologically

and functionally diverse.

Lymphopoiesis can be divided into antigen-independent and antigen

dependent lymphopoiesis.

22

Antigen independent lymphopoiesis occurs in the primary lymphoid tissue

(bone marrow, thymus, fetal liver, yolk sac). It begins with the committed lymphoid

stem cell and results in the formation of immunocompetent T and B lymphocytes

(nicknamed as ‘virgin lymphocytes’)

Antigen dependent lymphopoiesis occurs in secondary lymphoid organs (adult

bone marrow, spleen, lymph nodes, gut associated lymphoid tissue), and it begins

with the antigenic stimulation of the immunocompetent T and B lymphocytes. It

results in the formation of effector T and B lymphocytes, which mediate immunity,

through the production of lymphokines by T-lymphocytes and antibodies by B-

lymphocytes.

MEGALOBLASTIC ANEMIA61

Megaloblastic anaemia is caused by deficiency of folate or cobalamin (Vit

B12). The peripheral blood is sometimes characterised by pancytopenia and an

increased mean corpuscular volume (MCV). The bone marrow has a prevalence of

large, early stage haematopoietic precursor cells. Cytogenetic studies have shown

increased chromosomal breakage in the bone marrow. In folate-deficient cells, it is

hypothesized that decreased levels of co-enzyme 5,10-methylene-tetrahydrofolate

inhibit the conversion of deoxyuridylate (dump) to thymidylate (dTMP). This results

in increased rate of uracil misincorporation into DNA. These mis-incorporated uracil

residues that are located near each other on opposing strands of DNA have the

potential to produce double stranded DNA breaks. Due to these DNA breaks,

induction of p53, a transcriptional factor with tumor suppressor activity takes place.

Along with p53, the protein product p21 also accumulates which results in cell cycle

23

arrest. This cell cycle arrest by p53 has been thought to contribute to its tumor-

suppressor function. The accumulation of p53 in haematopoietic cells has also been

associated with death by apoptosis.

To conclude,

1. Haematopoietic cells undergo apoptosis due to intracellular folate or vitamin B12

deficiency. Since the deficiency of the latter causes folate trap and folate cannot

be utilised, this results in increased uracil mis-incorporation into DNA and

increased p53 and p21 proteins.

2. The erythroblasts that survive this apoptotic process give rise to larger than normal

reticulocytes (macrocytes).

Pathophysiology of aplastic anaemia47

Aplastic anemia, the paradigm of bone marrow failure syndromes is most

simply defined as peripheral blood pancytopenia and a hypocellular marrow.

The etiologic hypothesis of aplastic anaemia has been referred to as the seed,

soil, worm and fertiliser hypothesis. For a viable ‘seed’ (haematopoietic stem cell) to

grow,it must be planted in good ‘soil’ (microenvironment of bone marrow) conducive

to growth, protected from antagonists ‘worms’ (cellular or humoral immuno-

suppression of haematopoiesis) and nourished with ‘fertilisers’ (growth factors).

Some of the proposed causes of aplastic anaemia include62

(1) Abnormalities of the haematopoietic stem cells:

(2) Abnormal haematopoietic microenvironment:

(3) Immune Mechanisms:

(i) Decrease in haematopoietic factors produced by monocytes and

lymphocytes

24

(ii) Damage by cytokines that suppress haematopoiesis

(iii) Suppression of haematopoiesis by cytotoxic T-cells (Killer T cells):

(iv) Suppression of haematopoiesis by natural killer (NK) cells

(v) Drugs and chemicals

(vi) Radiation

(vii) Viral infections

(viii) Paroxysmal nocturnal hemoglobinuria

HYPERSPLENISM59

Pancytopenia in hypersplenism is thought to be due to two important

mechanisms.

1) An abnormal sequestration of cells in the splenic pulp where they may be

phagocytosed and destroyed. This abnormality of splenic function is an

exaggeration of its normal role in the removal of aged red cells.

2) Spleen, by a hormonal mechanism, inhibits the release of mature cells from the

marrow and sometimes arrests the maturation of developing cells.

Edward R Eichner (1979) put forward some mechanisms in certain conditions

of hypersplenism.23

Diseases Most likely mechanism

1) Hairy cell leukaemia Retention of hairy cells

2) Cirrhosis and splenic vein thrombosis Increased pooling of blood cells

3) Felty’s syndrome Immune system work hypertrophy

4) Gaucher’s disease Increased pooling and flow induced

dilutional anemia

25

MYELODYSPLASTIC SYNDROMES16

The neoplastic transformation of the myeloid stem cell is the event that leads to

Myelodysplasia. It rarely transforms into acute lymphoblastic leukaemia (ALL)

suggesting that the MDS myeloid stem cell has lost its lymphopoietic potential. Genetic

mutations regulate this process and these alterations determine both the histology and the

clinical aspects of the disease.

MDS is characterized by peripheral pancytopenia despite a normo or

hypercellular bone marrow because of increased apoptosis of haematopoietic bone

marrow cells resulting in ineffective hematopoiesis. Apoptosis can be used as a

prognostic factor, as poor prognosis correlates significantly with higher apoptotic rates

causing severe pancytopenia. Inhibition of apoptotic mechanisms may induce leukaemic

transformation in MDS.63

LYMPHOMAS64

Pancytopenia in lymphomas 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 hematopoiesis through the lymphokines. Thus

the main pathogenetic mechanism of cytopenias is the cell mediated suppression of

normal hematopoiesis.

HIV65,66

The wide ranging clinical and pathological changes in HIV patients are both

fascinating and challenging to physicians and pathologists.

26

Hematological abnormalities are well recognised in HIV diseases and result

from diverse influences on hematopoietic tissue changes in the peripheral blood and

bone marrow which may reflect disease elsewhere in the body and resist the treatment

for that disease or may reflect an attempt to attack the virus itself or may seem to be

an isolated hematological disorder.

In virtually all patients with advanced AIDS pancytopenia is the rule. The

causes are heterogeneous and commonly iatrogenic or multifactorial. The most

consistent hematopoietic defects that occur in the seropositive patients as a result of

HIV-1 infection, Per se, includes regenerative bone marrow failure in which on

demand hematopoiesis is suppressed.

The occurrence of antibodies against blood cells in patients with HIV infection

can be due to

• 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.

• A third possibility is that HIV acts as the direct inducer of autoimmunity.

27

Blood and bone marrow findings in various conditions associated with

pancytopenia

1. MEGALOBLASTIC ANAEMIA47

In megaloblastic anaemia, peripheral smear may show pancytopenia.

Ovalomacrocytes, usually with considerable anisopoikilocytosis, is the main feature.

Mean corpuscular volume (MCV) is more than 100 fl. In others, MCV may be normal

due to excessive fragmentation of red cells. Polychromatophilic cells are reduced.

Reticulocyte count may be less than 1%. The leucocyte count is reduced due to

reduction in the number of neutrophils and lymphocytes. Hypersegmented neutrophils

are usually seen. A minimum of five percent of five lobed or a single six lobed

neutrophil is considered significant. Thrombocytes are reduced in number, but rarely

cause hemorrhagic complications. Macrocytosis and hypersegmented neutrophils

occurring together strongly suggests megaloblastic haematopoiesis.

Bone marrow

Megaloblast is a designation first applied by Ehrlich to the abnormal

erythrocyte precursors found in the bone marrow of patients with pernicious anemia.

These are known to be the morphologic expressions of a biochemical abnormality and

retarded DNA synthesis. The aspirate is hypercellular. The M: E ratio is normal or

reduced and there is an accumulation of primitive cells due to selective death of more

mature forms. The most characteristic finding is dissociation between nuclear and

cytoplasmic development in the erythroblasts, with the nucleus maintaining a

primitive appearance despite maturation and hemoglobinization of the cytoplasm.

Fully hemoglobinized (orthochromatic) erythroblasts, which retain the nuclei, may be

28

seen. The nucleus of the megaloblast has an open, fine and lacy appearance: the cells

are larger than normoblasts and an increased number of cells with eccentric lobulated

nuclei or nuclear fragments may be present. Mitoses and dying cells are more

frequent than normal. Giant and abnormally shaped metamyelocytes and enlarged

hyperpolypoid megakaryocytes are characteristic.

2. APLASTIC ANAEMIA16

Peripheral smear

Patients with aplastic anemia have a variable degree of pancytopenia. The

haemoglobin percentage is reduced which is usually less than 7 g%. Reticulocyte

index is low and a retic count of <1% which may be even zero despite high levels of

erythropoietin may be seen. Red blood cells show moderate to marked anisocytosis

and poikilocytosis.

Anaemia is usually normocytic and normochromic but sometimes macrocytic.

The leucocyte and platelet counts are low. Neutrophils are more predominantly

affected than the lymphocytes. Significant qualitative changes of red cell, leucocyte

or platelet morphology are not features of classic acquired aplastic anemia.

Bone marrow

The marrow aspirate typically contains numerous spicules with empty fat

filled spaces with relatively few hematopoietic cells. Lymphocytes, plasma cells,

macrophages and mast cells may be prominent. A marrow biopsy is essential to

confirm the overall hypocellularity.

29

Criteria as suggested by International Aplastic anaemia study group

Site Findings

i. Neutrophils - <500/mm3

ii. Platelets - <20000 cells/cumm Blood

iii. Reticulocyte - <1% (corrected)

i. Severe hypocellularity

Bone marrow ii. Moderate hypocellularity with <30% residual cells representing haematopoietic cells.

Severe aplasia is defined by any two or three peripheral blood criteria and

either marrow criterion.

3. HYPERSPLENISM59

Peripheral Smear

There is nothing specifically diagnostic about the peripheral blood. Anemia is

usually normocytic and normochromic. Marked anisopoikilocytosis is rare. In a few

cases, reticulocytosis is present. Leucopenia is primarily due to neutropenia, but in

severe cases, all types of white cells are reduced in the blood. The white cell count is

frequently not sufficiently reduced to cause symptoms. The total white cell count

ranges from 1000 cells/cumm to 4000 cells/cumm. A moderate thrombocytopenia is

usual. The value ranges from 1,00,000 cells/cumm to 50,000 cells/cumm.

Bone marrow

The picture is either of normal cellularity or hypercellularity. Erythropoiesis is

usually normoblastic. The disease process which has been responsible for the

enlargement of the spleen may infiltrate it. In some cases, white cell precursors show

30

an arrest at the myelocyte or metamyelocyte stage. The megakaryocytic cells may

also show maturation arrest.

4. MYELODYSPLASTIC SYNDROMES16 (MDS)

Morphological abnormalities in MDS

Lineage Peripheral blood Bone marrow 1. Erythroid Oval macrocytes, acanthocytes,

elliptocytes, hypochromic fragments, basophilic stippling, nucleated red blood cells.

Ringed sideroblasts, megaloblasts, vacuolated cytoplasm, nuclear fragmentation hyperlobulation, Multinuclearity and inter nuclear bridging.

2. Granulocytic Nuclear hypolobulation nuclear hyper segmentation, ringed nuclei, coarse chromatin clumping, cytoplasmic hypo or agranularity

Loss of primary and secondary granules, increased blasts

3. Eosinophilic Agranular cytoplasm, mixed eosinophilic and basophilic granulation

4. Monocytic Abnormal nuclear lobulation, agranular cytoplasm

5. Megakaryocytic Agranular or giant platelets Micromegakaryocytes, large mono or binucleate

FAB Classification/ criteria for MDS (1982)

Subtype Abbreviation Peripheral blood Bone marrow Refractory anaemia RA < 1 % blasts <5% blasts Refractory anaemia with ringed sideroblasts

(RARS) < 1 % blasts <5% blasts, ≥ 15% ringed sideroblasts

Refractory anaemia with excess blasts

(RAEB) < 5 % blasts Blasts 5-20%

RAEB in transformation

(RAEBt) >5 % blasts Blasts 20-30% or Auer rods

Chronic myelomonocytic leukaemia

CMML Monocytes >1000cells/cumm

Any of the foregoing

Acute myeloid leukemia

AML Blasts >30%

31

The proposed WHO classification for MDS is

• Refractory anaemia: with ringed sideroblasts and without ringed sideroblasts.

• Refractory cytopenia (Myelodysplastic syndrome) with excess blasts.

• Refractory anaemia (Myelodysplastic syndrome) with excess blasts.

• 5q – syndrome

• Myelodysplastic syndrome, unclassifiable.

Chronic myelomonocytic leukaemia which is included under MDS in the FAB

classification is placed under the myelodysplastic/myeloproliferative disease along

with atypical chronic myelogenous leukaemia and juvenile myelomonocytic

leukaemia in the proposed WHO classification.

5. LEUKAEMIAS16

Peripheral Smear

In addition to pancytopenia, the smear shows a few immature blast cells. A

film made from the buffy coat may enable the cells to be detected more easily.

Bone marrow

Fragments in the aspirated marrow are usually fleshy and numerous. A bloody

tap is not uncommon and occasionally a dry tap occurs. Blast cells are the

predominant cells comprising 30-95% of the total marrow cells.

ACUTE MYELOID LEUKEMIA

Velpeau in 1827 reported the first accurate description of a case of leukemia in

a patient suffering from fever, weakness, urinary stones and massive hepatomegaly.

Bennet (1845) introduced the term Leukocythemia for the appearance of blood in

32

patients who died with enlarged spleen and changes in the color and consistency of

the blood. Virchow introduced the term “Leukemia” for similar cases. Ebstein (1889)

introduced the term “acute leukaemia” to describe a rapidly fatal illness that failed to

respond to available therapy. Neumann (1869) proposed the term myeloid for WBCs

as they originated from bone marrow and not spleen. Mosler (1879) described

aspiration of bone marrow as a means of diagnosing leukaemia. Naegeli (1900)

described myeloblast and divided leukemias into myelocytic or lymphocytic

categories.

Peripheral Smear

Most patients present with pancytopenia and circulating blasts. The WBC

count may range from 1000-2 lakh cells/cumm. In 10% of patients who present with

modest thrombocytopenia, low grade anemia and normal WBC count without

circulating blasts may be seen. A bone marrow examination is required to make the

diagnosis. Auer rods in the blasts are virtually pathognomic of acute myeloid

leukemia.

Bone marrow

Aspirate is hypercellular with absent or decreased megakaryocytes. However

in the elderly, it can be normo or hypocellular. Dysplastic myeloid and erythroid

maturation may be noted. Myeloid precursors may be morphologically bizarre with

asynchronous granulation.

ACUTE LYMPHOBLASTIC LEUKAEMIA16 (ALL)

By 1913, leukemia could be classified as acute or chronic and lymphatic or

myelogenous leukemia.

33

Blood

Anemia is usually normocytic normochromic and thrombocytopenia is

extremely common. Leucocyte count ranges from <5000 to >1 lakh/cumm. Peripheral

smear reveals leukemic lymphoblasts in 92% of patients.

Bone Marrow

Definitive diagnosis of ALL requires bone marrow examination which is

usually hypercellular and infiltrated by leukemic lymphoblasts. Presence of >5%

lymphoblasts cells confirms the diagnosis. In adults bone marrow cannot be aspirated

and a biopsy must be performed. Dry taps are due to densely packed blast cells,

fibrosis or improper technique.

HAIRY CELL LEUKAEMIA

Peripheral smear

The recognition of typical hairy cells in peripheral blood films is useful in

establishing the diagnosis. Hairy cells are large, twice the size of a normal

lymphocyte, and have abundant cytoplasm which is characteristically villous in its

outline. The nuclear chromatin is smooth and no nucleoli are seen. These cells are

Tartarate resistant acid phosphatase positive (TRAP).

Bone marrow

Aspirates are unsuccessful and a trephine biopsy is required. The infiltration is

interstitial. A typical feature is the loose arrangement of the cellular infiltrate, leaving

plenty of space between the cells, often with a clear zone around each cell which is

unique to this condition. The hairy cells are CD11C, CD25, HC2 and CD103 positive.

34

MULTIPLE MYELOMA (MM)67

Blood

Most patients with MM develop anemia of usually moderate severity.

Normocytic normochromic blood picture with rouleaux formation and a low

reticulocyte count is seen. The macrocytic indices may be the result of red cell

agglutination. The ESR is high. The leucocyte count is usually normal or reduced

with a mild neutropenia and an increase in plasma cells. The platelet count is usually

normal or low.

Bone marrow

Myeloma cells are characteristically present in all the patients. The Myeloma

cell is moderately large (15-30µm) with a round or ovoid, eccentrically placed

nucleus and one or two nucleoli. The bone marrow is infiltrated with an excess of

plasma cells (>5%) in approximately 90% of cases.

Criteria for the diagnosis of Multiple Myeloma

Major

• Plasmacytomas on tissue biopsy

• Marrow plasmacytosis with > 30% plasma cells.

• Monoclonal globulin spike on serum electrophoresis (> 3.5g/dl for IgG or > 2g/dl

for IgM)

Minor

• Marrow plasmacytosis 10-30%

• Lytic bone lesions

• Normal IgM < 0.05 g/dl ,IgA< 0.1g/dl or IgG < 0.6g/dl

35

• Monoclonal globulin spike is present but is less than the levels defined above.

• Minimum of 1 major + 1 minor or 3 minor criteria are required to diagnose the

condition.

6. INFECTIONS16

Bacterial, fungal, protozoal and viral infectious agents are sometimes

associated with pancytopenia, which ranges from mild depression in blood counts to

severe trilineage aplasia. It may be caused by direct toxic effects leading to bone

marrow necrosis, bone marrow replacement (myelophthisis), inhibitory effects of

inflammatory mediators, or activation of the reticuloendothelial system leading to

hemophagocytic syndrome. Persistent congestive splenomegaly as a result of

infection can also cause pancytopenia.

Viral infections associated with pancytopenia include those with arboviruses

such as dengue, which cause transient effects, and suspected agents such as Non A to

Non C hepatitis which appear to be associated with more durable aplastic anemia.

Blood

Anemia is usually normocytic, normochromic or mildly microcytic and

hypochromic with a low reticulocyte count. Changes in leucocyte parameters include

cellular morphology and number. Early changes in infection include an increase in

band forms and a greater shift to immaturity (left shift) which occurs in severe

infections. Toxic granulations, cytoplasmic vacuolization and Dohle bodies are seen.

Thrombocytopenia in the absence of Disseminated intravascular coagulation

(DIC) may be seen in septicaemia associated with bacterial, fungal, or viral

36

infections, the mechanism being suppression of platelet production, increased platelet

utilisation and immune phenomena.

Bone Marrow

The marrow is often hypocellular. Granulopoiesis and erythropoiesis in

particular may be markedly decreased. The number of megakaryocytes in the marrow

is normal or reduced slightly.

7. HIV INFECTION68

Peripheral smear findings include leucopenia which is a common feature and

includes lymphopenia as well as granulocytopenia. A shift to the left of the

granulocyte series is a common finding. Anaemia and thrombocytopenia are seen.

Pancytopenia is a common finding in advanced AIDS (group IV).

Features of bone marrow in HIV disease

I. Dysplasia 70% of marrows show some dysplastic features i. Erythroid dysplasia – 60%

Binucleated, multinucleated megaloblastic erythroblasts. Abnormal sideroblasts including ringed sideroblasts ii. Granulocytic dysplasia – 20-30%. Apparent at all stages of maturation with megaloblastic

change, nuclear abnormalities, and Pelger cells reflecting dysfunctional nuclear maturation.

iii. Megakaryocytic dysplasia – nuclear. Hypolobulation and micromegakaryocytes

II. Cellularity Increased in over 50%, hypocellular in 15% III. Fibrosis Seen in 20-50% of trephines and may make aspiration difficult. IV. Reticulo-

Endothelial Common manifestations of chronic disease or concurrent infection.

V. Histiocytes Increased and may show hemophagocytosis VI. Plasma Cells Increased and may be atypical VII.Opportunistic

infections Culture or examination of slides may show acid fast bacilli, histoplasma, leishmania, pneumocystis, Cryptococcus

37

Tilak and Jain10 (1999) in their analysis of 77 cases of pancytopenia, found

megaloblastic anemia (68%) as the commonest cause followed by aplastic

anemia (7.70%) Other uncommon causes were drug induced agranulocytosis,

hemophagocytic syndrome and Waldenstrom’s macroglobinemia.

Savage et al.69 (1999) studied 134 cases of pancytopenia and noted

megaloblastic anemia as the most common cause. Other causes were aplastic anemia,

acute leukemia, AIDS and hypersplenism.

Kumar et al.70 (1999) in their analysis of 166 cases of pancytopenia, found

hypoplastic anemia (29.51%), followed by megaloblastic anemia (22.3%) as

the common causes. Other causes were aleukemic leukemia/lymphoma and

hypersplenism.

Khodke et al. (2000), studied 50 cases of pancytopenia cases and observed

megaloblastic anemia (44%), followed by hypoplastic anemia (14%) as the common

causes. Other causes noted were kala azar, multiple myeloma, myelodysplastic

syndrome, tuberculosis and acute leukemia.

Naeem Khan et al.71 (2001) studied 30 cases of pancytopenia and found

aplastic anemia as the most common cause (20%). Other causes were megaloblastic

anemia (16.7%), acute leukemia, MDS, PNH, SLE, falciparum malaria, dengue fever,

multiple myeloma, Hodgkin’s disease and disseminated malignancies.

Osama et al.72 (2002) in their study of 100 pancytopenia cases found

megaloblastic anemia as the most common cause. Other etiological factors were

hemolytic anemia, septicaemia, hypersplenism, aplastic anemia, myelodysplasia and

viral fever.

38

Muzzaarat et al.73 (2004) in their study of 189 pancytopenia cases found

aplastic anemia as the most common cause followed by megaloblastic anemia. Other

etiological factors were hypersplenism and acute leukemia.

Jha et al.3 (2008) in their study of 148 cases of pancytopenia found aplastic

anemia (29%) as the most common cause followed by megaloblastic anemia

(23.64%). Other etiological factors were hematological malignancies, metastatic

neuroblastoma, plasma cell myeloma, Non Hodgkin’s lymphoma and leishmaniasis.

39

METHODOLOGY

Clinical history recording and examination of all the identified cases of

pancytopenia were done as per the proforma.

Two ml of anticoagulated blood was collected for complete hemogram.

Following investigations were done.

Investigation Method

Bleeding time Ivy’s method

Clotting time Tube method

Hemoglobin percentage Sysmex kx 21

Total leucocyte count Sysmex kx 21

RBC Count Sysmex kx 21

Platelet count Sysmex kx 21

Packed cell volume Sysmex kx 21

Red cell indices Sysmex kx 21

Reticulocyte count New methylene blue

HIV, HBsAg ELISA and Tridot Method The peripheral smear was studied after staining with Leishman’s stain.

Special stains – Periodic acid Schiff reagent stain, Myeloperoxidase, Sudan

black and Perls’ stains were used wherever indicated.

Bone marrow aspiration

This was done in all the patients to identify the etiology. An informed consent

was obtained. A Jamshidi needle was used to aspirate material from the Posterior iliac

40

crest in adults. Local anaesthetic infilitration was done after administering a test dose.

Sterile precautions were observed. The needle and the stillette were placed in position

and the cap was closed. After piercing the skin and subcutaneous tissue, the

periosteum and cortex were pierced with a drilling action. Once in the marrow cavity,

the stillette was removed and 0.2-0.3 ml of marrow fluid was aspirated with a sterile

disposable 10 ml syringe. The aspirate was transferred to a set of slides and smeared.

The needle was withdrawn and a tincture benzoin seal applied. Slides were stained

with Leishman’s stain. In case of failure, bone marrow aspirations were done at

different sites.

Bone marrow biopsy

Biopsy whenever required was done along with aspiration in the same sitting.

Following aspiration, the stillette was withdrawn from the needle and then the cap

was closed. Now it was further pushed into the cavity by rotating movements for

about 0.5-1 cm. This process captures the marrow core sample within the needle. The

needle was then withdrawn in the reverse rotating direction. A wire probe was

inserted at the hub of needle on to a sterile gauge. The specimen was fixed in 10%

formalin overnight and decalcified with 6% EDTA for 72 hours. Then it was

processed similar to histopathological sample and H&E sections were studied. Special

stains like PAS and reticulin were done whenever necessary.

41

Figure 1: Materials used for Bone Marrow aspiration and Biopsy

Figure 2: Bone Marrow aspirate particles on a glass slide

42

Figure 3: Normal bone marrow. (Leishman’s stain 10x X 10x)

Figure 4: Bone marrow aspiration smear showing Erythroid Hyperplasia

(Leishman’s stain 10x X 10x)

43

Figure 5: Megaloblastic anemia. Peripheral smear showing hypersegmented

neutrophils and macroovalocytes. Leishman’s stain – 10x X 100x

Figure 6: Megaloblastic anemia. Bone marrow aspiration smear showing hypercellular marrow. Leishman’s stain – 10x X 10x

44

Figure 7: Megaloblastic anemia. Bone marrow aspiration smear showing megaloblasts with open chromatin. Leishman’s stain – 10x X 100x

Figure 8: Megaloblastic anemia. Bone marrow aspiration smear showing giant band form, mitosis and metamyelocyte. Leishman’s stain- 10x X 100x

45

Figure 9: Aplastic anemia. Bone marrow aspiration smear showing increase in fat cells. Leishman’s stain- 10x X 10x

Figure 10: Aplastic anemia. Bone marrow aspiration smear showing increase in

fat cells. Leishman’s stain- 10x X 40x

46

Figure 11: Aplastic anemia. Bone marrow trephine biopsy showing increase in

fat cells. H & E Stain- 10x X 40x

Figure 12: AML. Bone marrow aspiration smear showing myeloblasts.

Leishman’s stain- 10x X 40x

47

Figure 13: AML. Bone marrow aspiration smear showing myeloblasts positive

for MPO.-10x X 100x

Figure 14: MDS. Bone marrow aspiration smear showing dyserythropoiesis and

blasts. Leishman’s stain- 10x X 100x

48

Figure 15: MDS. Bone marrow aspiration smear showing dyserythropoiesis and mitosis. Leishman’s stain- 10x X 100x

49

RESULTS

Fifty patients with a hematological diagnosis of pancytopenia were studied

during the period, November 2008 to May 2010, in the Department of Pathology,

Mysore Medical College and Research Institute, Mysore.

The following data was recorded and analysed.

Age and sex distribution of patients with pancytopenia

Table 1: Age and sex distribution of patients with pancytopenia

Age group (years) Female Male Total no. of

cases Percentage

18-20 1 2 3 6

21-30 3 4 7 14

31-40 3 8 11 22

41-50 3 11 14 28

51-60 6 7 13 26

61-70 0 2 2 4

Total 16 34 50 100

χ2=3.352;p<.10;df=5 Most of the patients were in the age group of 41-60 years (54%) and least

occurrence was seen in the age group of 61-70 years (4%). The sex distribution of

pancytopenia showed a male preponderance. The male to female ratio was 2.1:1.

50

Figure 16: Age and sex distribution of patients with pancytopenia

1

2

3

4

3

8

3

11

67

0

2

0

2

4

6

8

10

12

Num

ber o

f pat

ient

s

18-20 21-30 31-40 41-50 51-60 61-70

Age group (years) FemaleMale

Clinical features

Table 2: Clinical features

Symptoms No. of cases Percentage Chi-square and p-value

Generalised weakness 44 88 χ2=16.877;p<.000

Fever 19 38 χ2=1.461;p<.227

Bleeding 3 65 χ2=24.008;p<.000

Pain abdomen 23 46 χ2=0.160;p<.689

Abdominal distension 41 82 χ2=11.408;p<.001

Fever with rashes 5 10 χ2=19.048;p<.000 Generalised weakness (88%) was the commonest symptom in pancytopenic

patients, followed by fever (38%), bleeding manifestations (6%), pain abdomen

(46%), abdominal distension (41%) and fever with rashes (10%).

51

Hematological Data

1. Hemoglobin percentage

Table 3: Range of hemoglobin in patients with pancytopenia

Hemoglobin percentage No. of cases Percentage

1 to 3 5 10

3.1 to 5 10 20

5.1 to 7 25 50

7.1 to 10 10 20

Total 50 100 χ2= 8.092;p<.044(S) Figure 17: Range of hemoglobin in patients with pancytopenia

10(20%)

5(10%)10(20%)

25(50%)

1 to 33.1 to 55.1 to 77.1 to 10

The hemoglobin percentage varied from 1 gm% to 10 gm%. Majority (50%)

of the patients had hemoglobin ranging from 5.1-7 g%. 10% of the patients had

hemoglobin values between 1% and 3%.

52

2. Total leukocyte count

Table 4: Range of leukocyte count in patients with pancytopenia

Leukocyte count (cells/cumm) No. of cases Percentage

500-1000 3 6

1100-2000 11 22

2100-3000 14 28

3100-4000 22 44

Total 50 100

χ2 = 8.732;p<.033(S) Figure 18: Range of leukocyte count in patients with pancytopenia

3

11

14

22

0

5

10

15

20

25

Num

ber o

f pat

ient

s

500-1000 1100-2000 2100-3000 3100-4000

Leukocyte count (cells/cumm)

The total leukocytic count was in the range of 500-4000 cells/cumm. Most

(44%) of the patients had values in the range of 3100-4000 cells/cumm. 6% of the

patients had values in between 500 and 1000 cells/cumm.

53

3. Platelet count

Table 5: Range of platelet count in patients with pancytopenia

Platelet count (cells/cumm) No. of cases Percentage

4000-25000 2 4

26000-50000 9 18

51000-75000 20 40

76000-1,00,000 12 24

101000-1,50,000 7 14

Total 50 100

χ2= 9.431;p<.051(NS) Figure 19: Range of platelet count in patients with pancytopenia

2

9

20

12

7

02468

101214161820

Num

ber o

f pat

ient

s

4000-25000 26000-50000 51000-75000 76000-1,00,000

101000-1,50,000

Platelet count (cells/cumm)

The range of platelet count varied from 4000-1,50,000 cells/cumm. Most

(40%) patients had platelet counts in the range of 51,000-75,000 cells/cumm.

54

4. Reticulocyte count

Table 6: Range of reticulocyte count in patients with pancytopenia

Reticulocyte count No. of cases Percentage

0.1-2 41 82

2.1-4 5 10

4.1-6 2 4

6.1-8 0 0

8.1-20 2 4

Total 50 100

χ2= 41.176; p<.000(NS) Figure 20: Range of reticulocyte count in patients with pancytopenia

2(4%)

41(82%)

2(4%)5(10%)

0.1-22.1-44.1-66.1-88.1-20

The reticulocyte count varied from 0.1-20%. Majority (82%) of the patients

had reticulocyte count in the range of 0.1-2%, 4% of them had a value in between

8.1 and 20%.

55

Cellularity bone marrow

Table 7: Bone marrow cellularity in patients with pancytopenia

Type of cellularity No. of cases Percentage

Hypercellular 38 76

Hypocellular 5 10

Normocellular 7 14

Total 50 100

χ2= 18.722;p<.000 Bone marrow aspirate in the present study of pancytopenia showed the

following types of cellularity.

a. Hypocellularity – 10%

b. Hypercellularity – 76%

c. Normocellular – 14%

Further analysis of data was done under the following headings:

1. Pancytopenia associated with hypocellular marrow.

2. Pancytopenia associated with hypercellular marrow and normocellular marrow

a) Pancytopenia associated with megaloblastic anemia

b) Pancytopenia associated with nutritional anemia

c) Pancytopenia associated with hypersplenism

d) Pancytopenia associated with malignant diseases and myelodysplastic

syndromes

i) Acute myeloblastic leukemia

ii) Myelodysplastic syndrome

e) Others

56

I. PANCYTOPENIA ASSOCIATED WITH HYPOCELLULAR MARROW

In the present study, 5 out of 50 patients had hypocellular marrow.

1. Age and sex distribution

Table 8: Age and sex distribution of patients with aplastic anemia

Age (years) Female Male Total no. of cases Percentage

18-20 0 0 0 0 21-30 0 0 0 0 31-40 0 1 1 20 41-50 1 2 3 60 51-60 1 0 1 20 61-70 0 0 0 0 Total 2 3 5 100

χ2= 2.222;p<.10;df=2

Aplastic anemia showed a peak incidence (60%) in the age group

(41-50 years). The least incidence (20%) was seen in the age group of 31-40 years.

Aplastic anemia was more common in males (60%). The male to female ratio of

incidence was 1.5:1.


Table 9: Range of hemoglobin in patients with aplastic anemia Hemoglobin percentage No. of cases Percentage

1-3 0 0 3.1-5 2 40 5.1-7 2 40

7.1-10 1 20 Total 5 100

χ2= 0.244;p<.885

The haemoglobin percentage varied from 3.1-10 gm%. Most patients (40%)

had hemoglobin values in the range of 5.1-7 g%. 20% of patients were seen to have

hemoglobin values in the range of 7.1-10 g%.

57


Table 10: Range of leukocyte count in patients with aplastic anemia


500-1000 0 0

1100-2000 2 40

2100-3000 1 20

3100-4000 2 40

Total 5 100

χ2= 0.244;p<.885

The total leukocyte count ranged from 1100-4000 cells/cumm. Most patients

(40%) had a value in the range of 1100-2000 cells/cumm and 3100-4000 cells/cumm.

4. Platelet count

Table 11: Range of platelet count in patients with aplastic anemia


4000-25000 0 0

26000-50000 0 0

51000-75000 2 40

76000-1,00,000 2 40

101000-1,50,000 1 20

Total 5 100

χ2= 0.244;p<.885

The platelet count was in the range of 51,000-1,50,000 cells/cumm. Most of

them (80%) had platelet values in the range of 51,000-1,00,000 cells/cumm.

58


Table 12: Range of reticulocyte count in patients with aplastic anemia


0.1-0.5 2 40

0.6-1.0 2 40

1.1-1.5 1 20

1.6-2 0 0

Total 5 100

χ2= 0.244;p<.885

The reticulocyte count was in the range of 0.1-1.5%. Majority of the patients

(80%) had values in the range of 0.1-1.0%.

6. Blood picture

Most of the patients (64.2%) had normocytic normochromic erythrocytes.

Some (35.8%) showed macrocytosis. 42.8% of them had relative neutrophilia while

the others had relative lymphocytosis. The erythrocyte sedimentation rate was

increased in most of the patients and ranged from 28-140 mm/hr.

7. Bone marrow

The bone marrow was hypocellular and the aspirate was mostly composed of

fat cells. Other precursors appeared normal. There was a relative increase in the

number of plasma cells and lymphocytes.

59

II. PANCYTOPENIA WITH HYPERCELLULAR AND NORMOCELLULAR

MARROW

Table 13: Pancytopenia with hypercellular and normocellular marrow

Total no. of cases Hypercellular Normocellular

Etiology No. % No. % No. %

Megaloblastic anemia 22 100 18 81.8 4 18.2

Nutritional anemia 10 100 9 90 1 10

Hypersplenism 6 100 4 66.7 2 33.3

Leukemia 3 100 3 100 0 0

Myelodysplastic syndrome 1 100 1 100 0 0

Dengue 2 100 2 100 0 0

Hemolytic anemia 1 100 1 100 0 0

χ2=3.084;p<.10;df=6

Hypercellular bone marrow was observed in 38 patients and it was normo-

cellular in 7 patients.

Most common etiology noted was megaloblastic anemia (48.9%), followed by

nutritional anemia (22.2%), hypersplenism (13.3%), leukemia (6.7%),

myelodysplastic syndromes (2.2%) and others (6.6%). Normocellular marrow was

seen in (8%) megaloblastic anemia, (4%) hypersplenism and 2% in nutritional

anemia.

A. PANCYTOPENIA WITH MEGALOBLASTIC ANEMIA

In the present study, 22 cases of megaloblastic anemia were seen. It

constituted 47.4% of cases with hypercellular marrow, and 44% of all patients with

pancytopenia.

60


Table 14: Age and sex distribution of patients with megaloblastic anemia


18-20 0 0 0 0

21-30 1 2 3 13.6

31-40 2 5 7 31.8

41-50 0 7 7 31.8

51-60 2 2 4 18.2

61-70 0 1 1 4.6

Total 5 17 22 100 χ2=4.375;p<.10;df=4 Megaloblastic anemia was seen to occur in the age group ranging from

21-70 years. Majority of the patients were seen in the age group of 31-50 years

(63.6%). There were male preponderance and the male to female ratio was 2.4:1.


Table 15: Range of hemoglobin in patients with megaloblastic anemia


1-3 2 9.1

3.1-5 2 9.1

5.1-7 15 68.2

7.1-10 3 13.6

Total 22 100 χ2=7.994;p<.046(S) Hemoglobin percentage varied from 2-10 g%. Majority of the patients (68.2%)

had values in the range of 5.1-7 gm%.

61


Table 16: Range of leukocyte count in patients with megaloblastic anemia


500-1000 1 4.6

1100-2000 3 13.6

2100-3000 9 40.9

3100-4000 9 40.9

Total 22 100

χ2=5.369;p<.147

The total leukocyte count ranged from 500-4000 cells/cumm. Majority of the

patients (81.8%) had a leukocyte count in the range of 2100-4000 cells/cumm. 4.6%

of patients had values ranging from 500-1000 cells/cumm.

4. Platelet count

Table 17: Range of platelet count in patients with megaloblastic anemia


4000-25000 0 0

260000-50000 2 9.1

51000-75000 9 40.9

76000-1,00,000 8 36.4

101000-1,50,000 3 13.6

Total 22 100

χ2=3.534;p<.316

The platelet count varied from 26,000-1,50,000 cells/cumm. Most of the

patients (40.9%) had a platelet count in the range of 51,000-75,000 cells/cumm. 9.1%

patients had values in the range of 26,000-50,000 cells/cumm.

62


Table 18: Range of reticulocyte count in patients with megaloblastic anemia


0.1-2 17 77.3

2.1-4 4 18.2

4.1-6 1 4.5

6.1-8 0 0

8.1-20 0 0

Total 22 100 χ2=9.467;p<.009

The reticulocyte count varied from 0.1-6%. Most of them (77.3%) had

reticulocyte count in the range of 0.1-2%.

6. Peripheral smear findings

Macroovalocytosis with a considerable degree of anisopoikilocytosis were the

main features. Mean corpuscular volume was more than 100 fl in (57.5%) of patients.

Dimorphic blood picture was seen in 10 patients (30%).

Hypersegmented neutrophils were seen in most of the patients. Basophilic

stippling and cabot rings were present. Platelets were reduced in number in all the

cases.

7. Bone marrow

The bone marrow was hypercellular with a reduction of fat cells in most of

the patients (81.8%). Four patients (18.2%) had a normocellular marrow. Erythroid

hyperplasia with megaloblastic maturation and reversal of M:E ratio was seen in all

the patients. Megakaryopoiesis was normal in 63.6%, decreased in 18.2% and

increased in 18.2% of patients.

63

B. PANCYTOPENIA WITH NUTRITIONAL ANEMIA

In the present study, 10 patients (20%) of pancytopenia were observed to

have nutritional anemia as the etiology, which constituted 23.7% of cases with

hypercellular marrow.


Table 19: Age and sex distribution of patients in nutritional anemia


18-20 1 1 2 20 21-30 0 1 1 10 31-40 0 1 1 10 41-50 1 1 2 20 51-60 1 3 4 40 61-70 0 0 0 0 Total 3 7 10 100

χ2=1.667;p<.025;df=4

Nutritional anemia was seen in the age group of 18-60 years. Majority of the

patients (40%) were in the age group of 51-60 years. 20% of them were in the age

group of 21-40 years. There was a male predominance and the male to female ratio of

incidence was 2.3:1.


Table 20: Range of hemoglobin in patients with nutritional anemia Hemoglobin percentage No. of cases Percentage

1-3 1 10 3.1-5 5 50 5.1-7 3 30

7.1-10 1 10 Total 10 100

χ2=1.954;p<.584

Hemoglobin percentage varied from 2.3-7.8%. Majority of the patients (50%)

had haemoglobin in the range of 3.1-5 gm%.

64


Table 21: Range of leukocyte count in patients with nutritional anemia


500-1000 1 10

1100-2000 0 0

2100-3000 3 30

3100-4000 6 60

Total 10 100

χ2=1.953;p<.377

The total leukocyte count ranged from 1000-4000 cells/cumm. Most of the

patients (60%) had a value in the range of 3100-4000 cells/cumm. 10% of the patients

had a count of 500-1000 cells/cumm.

4. Platelet count

Table 22: Range of platelet count in patients with nutritional anemia


4000-25000 2 20

26000-50000 3 30

51000-75000 4 40

76000-1,00,000 0 0

101000-1,50,000 1 10

Total 10 100

χ2=0.990;p<.804

The platelet count varied from 5000-1,40,000 cells/cumm. Most of the patients

(40%) had a value in between 51,000 and 75,000 cells/cumm.

65


Table 23: Range of reticulocyte count in patients with nutritional anemia


0.1-2 5 50

2.1-4 2 20

4.1-6 2 20

6.1-8 1 10

8.1-20 0 0

Total 10 100

χ2=1.414;p<.702

The reticulocyte count varied from 0.1-8%. In majority of the patients (50%)

the values ranged from 0.1-2%.

6. Peripheral smear

Most of the patients had normocytic normochronic anemia. In two patients had

microcytic hypochromic anemia, MCV was in the range of 65.6-108 fl.

MCHC was in the range of 26-34.7% and MCH was in the range of

18-38.9 pg.

7. Bone marrow

Nine patients (90%) had hypercellular marrow with a reversal of M:E ratio.

Erythroid hyperplasia along with both megaloblastic and micronormoblastic

maturation was observed in all the patients. Leucopoiesis was normal.

Megakaryocytes were either normal or increased. Only two patients had decreased

megakaryopoiesis.

66

C. PANCYTOPENIA WITH HYPERSPLENISM

In the present study, 6 patients of hypersplenism were seen, which constituted

10.5% of cases with hypercellular marrow and 12% of all patients with pancytopenia.


Table 24: Age and sex distribution in patients with hypersplenism


18-20 0 0 0 0

21-30 0 0 0 0

31-40 0 0 0 0

41-50 1 1 2 33.3

51-60 1 2 3 50

61-70 0 1 1 16.7

Total 2 4 6 100 χ2=0.750;p<.10;df=2

Hypersplenism was seen to occur in the age group ranging from 41-70 years.

Majority of them were in the age group of 51-60 years. There was a male

preponderance and male to female ratio of incidence was 2:1.


Table 25: Range of hemoglobin in patients with hypersplenism Hemoglobin percentage No. of cases Percentage

1-3 0 0

3.1-5 1 16.7

5.1-7 2 33.3

7.1-10 3 50

Total 6 100 χ2=0.533;p<.766

Hemoglobin percentage varied from 3.8-10 gm%. Majority of the patients

(50%) had values in the range of 7.1-10 gm%.

67


Table 26: Range of leukocyte count in patients with hypersplenism


500-1000 0 0

1100-2000 3 50

2100-3000 1 16.7

3100-4000 2 33.3

Total 6 100

χ2=0.533;p<.766

The total leukocyte count ranged from 1700-3800 cells/cumm. Majority of the

patients (50%) had values in between 1100-2000 cells/cumm.

4. Platelet count

Table 27: Range of platelet count in patients with hypersplenism


4000-25000 0 0

26000-50000 1 16.7

51000-75000 2 33.3

76000-1,00,000 1 16.7

101000-1,50,000 2 33.3

Total 6 100

χ2=0.389;p<.943

The platelet count was in the range of 26,000-1,40,000 cells/cumm. Majority

(33.3%) of the patients had values in the range of 51,000-75,000 and 1,01,000-

1,50,000 cells/cumm.

68


Table 28: Range of reticulocyte count in patients with hypersplenism


0.1-0.5 0 0

0.6-1 3 50

1.1-1.5 2 33.3

1.6-2 1 16.7

Total 6 100

χ2=0.533;p<.766

The reticulocyte count ranged from 0.6-2%. Majority (50%) of the patients

had values in the range of 0.6-1%.

6. Peripheral blood

Most of the patients (60%) had normocytic normochromic anemia. 40% of

them had microcytic hypochromic anemia with severe degree of anisopoikilocytosis.

MCV was in the range of 56.8 fl – 94.6 fl.

7. Bone marrow

66.7% of patients had hypercellular marrow while the rest had normocellular

(33.3%). Erythroid hyperplasia was observed in 90% of patients. Leukopoiesis was

normal in 90% patients. Megakaryopoiesis was normal in 60% of patients, while it

was increased in the rest.

69

D. PANCYTOPENIA WITH MALIGNANT DISEASES,MYELODYSPLASTIC

SYNDROMES AND OTHERS

In the present study, 14% of the patients with pancytopenia had the following

etiology.

a) Malignant diseases – Acute myeloblastic leukaemia 6%

b) Myelodysplastic syndromes 2%

c) Dengue fever 4%

d) Hemolytic anemia 2%

Total 14% LEUKEMIA


Table 29: Age and sex distribution of patients with leukemia


18-20 0 0 0 0

21-30 0 1 1 33.3

31-40 1 1 2 66.7

41-50 0 0 0 0

51-60 0 0 0 0

61-70 0 0 0 0

Total 1 2 3 100

The age ranged from 21-40 years. Majority of them (66.7%) were in the age

group of 31-40 years. There was a male predominance. The male to female ratio of

incidence was 2:1.

70


Table 30: Range of hemoglobin in patients with leukemia


1-3 0 0

3.1-5 0 0

5.1-7 1 33.3

7.1-10 2 66.7

Total 3 100 χ2=0.139;p<.10;df=1

The hemoglobin percentage ranged from 5.1-9.8%. 66.7% of the patients had

values in between 7.1 and 10 gm%.


Table 31: Range of total leukocyte count in patients with leukemia

Total leukocyte count (cells/cumm) No. of cases Percentage

500-1000 0 0

1100-2000 1 33.3

2100-3000 0 0

3100-4000 2 66.7

Total 3 100

χ2=0.139;p<.10;df=1 The total leukocyte count varied from 1100-4000 cells/cumm. 66.7% of the

patients had values in between 3100-4000 cells/cumm.

71

4. Platelet count

Table 32: Range of platelet count in patients with leukemia


4000-25000 0 0

26000-50000 1 33.3

51000-75000 1 33.3

76000-1,00,000 1 33.4

101000-1,50,000 0 0

Total 3 100

χ2=0.000;p<1.000

Platelet count ranged from 26,000-89,000 cells/cumm. 33.4% of the patients

had values in between 76,000 and 1,00,000 cells/cumm.


Table 33: Range of reticulocyte count in patients with leukemia


0.1-0.5 0 0

0.6-1 2 66.7

1.1-1.5 0 0

1.6-2 1 33.3

Total 3 100

χ2=0.139;p<.10;df=1

The reticulocyte count ranged from 0.6-2%. 66.7% of the patients had a value

ranging from 0.6-1%.

72

6. Peripheral blood

These patients presented with peripheral pancytopenia. The erythrocytes were

normocytic normochromic. The leukocyte count was decreased with a presence of

immature cells. Myeloblasts with fine chromatin, 2-3 nucleoli and occasional Auer

rods were seen. Platelets were decreased.

7. Bone marrow

Bone marrow was hypercellular in 100% of the patients. Erythroid series were

decreased in 40% of the patients and increased in 60% patients. Myeloid hyperplasia

was observed with more than 30% blasts in all of them. Immunocytochemistry

demonstrated MPO positivity in them. Megakaryocytes are reduced.

MYELODYSPLASTIC SYNDROMES

One case of myelodysplastic syndrome was observed in the present study in a

female patient aged 55 years who had peripheral pancytopenia with a macrocytic type

of anemia.

The aspirate was hypercellular with erythroid hyperplasia and features of

dyserythropoiesis which included megaloblastic erythroblasts, nuclear fragmentation,

budding multiple nuclei and hyperlobulation. Dysmyelopoiesis with hypogranular

neutrophils and blasts were also observed. Large hypolobulated megakaryocytes were

seen.

DENGUE FEVER

Two cases of dengue fever in a 18 year old male and a 22 year old female

presented with pancytopenia. The total leukocyte count was 800 cells/cumm and

73

platelet count was 28,000 cells/cumm. Bone marrow was hypercellular with erythroid

hyperplasia and a reversal of M:E ratio.

HEMOLYTIC ANEMIA

One case of hemolytic anemia presented with pancytopenia. The patient was a

21 year old female. The peripheral blood showed features of hemolysis. Erythrocytes

showed moderate degree of anisopoikilocytosis with increased polychromatophilic

RBCs. The reticulocyte count was markedly increased.

The bone marrow was hypercellular with a reversal of M:E ratio. Erythroid

hyperplasia with normoblastic maturation was seen. Megakaryocytic hyperplasia was

also noted.

74

DISCUSSION

In the present study, pancytopenia was due to the following causes.

1. Aplastic anemia

2. Megaloblastic anemia

3. Nutritional anemia

4. Hypersplenism

5. Malignant disease – Leukemia

6. Myelodysplastic syndromes

7. Others

a) Dengue fever

b) Hemolytic anemia

In the present study, megaloblastic anemia (44%) was the commonest cause of

pancytopenia, followed by nutritional anemia (20%), hypersplenism (12%), aplastic

anemia (10%), malignant diseases (6%), myelodysplastic syndromes (2%) and others

(6%). Others included uncommon causes like Dengue fever (4%) and Hemolytic

anemia (2%).

In 1976, the study by Relief FP, Heyns AD18 revealed that bone marrow

failure (67.7%) was the commonest cause of pancytopenia and severe infections

(9.7%) was the second common cause.

In 1987, the International agranulocytosis and aplastic anemia study group5

found aplastic anemia in 52.7% and MDS in 10.5% of patients. Hossain MA et al.74

(1992) found aplastic anemia followed by chronic malaria and kalaazar to be the

commonest causes of pancytopenia.

75

Verma N, Dash S8 (1992) found aplastic anemia in 40.6% and megaloblastic

anemia in 23.26% of patients. Tilak V, Jain R10 (1998) found megaloblastic anemia

(68%) to be the commonest cause of pancytopenia followed by aplastic anaemia

(7.7%). Savage DG et al.69 (1999) found megaloblastic anemia to be the commonest

cause followed by aplastic anemia. Kumar et al.70 (1999) found hypoplastic anemia

(29.5%) to be the commonest cause followed by megaloblastic anemia.

Khodke et al.2 (2000) observed megaloblastic anemia (44%), followed by

hypoplastic anemia (14%) as the common causes of pancytopenia. Naeem Khan

et al.71 (2001) found aplastic anemia (20%) as the commonest cause of pancytopenia

followed by megaloblastic anemia (16.7%).

Osama72 found megaloblastic anemia (39%) as the commonest cause of

pancytopenia followed by hypersplenism (19%). Muzzarat Niazi73 (2004) found

aplastic anemia (38.3%) as the commonest anemia (24.7%). Mobina Ahsan Sodhy

(2005) found megaloblastic anemia (35.9%) followed by hypersplenism (16.3%) as

the common causes. Jha A et al.3 (2008) found hypoplastic bone marrow (29%)

followed by megaloblastic anemia (23.64%) as the common causes.

In the present study, megaloblastic anemia (44%) was the commonest cause of

pancytopenia followed by nutritional anemia (20%).

The commonest cause of pancytopenia, reported from various studies

throughout the world has been aplastic anemia.

76

This is in sharp contrast with the results of present study where the commonest

cause of pancytopenia was megaloblastic anemia. This seems to reflect the higher

prevalence of nutritional anemia in Indian subjects as well as in developing countries.

However similar results have been reported in studies from other Indian centres.

Table 34: Causes of pancytopenia in various studies

Sl. No. Study Country Year No.of

cases Commonest cause Second most common cause

1 Retief FP, Heyns AD

South Africa 1976 195 Bone marrow failure

(67.7%)

Severe infection (9.7%)

2

International agramilocytosis and aplastic anemia study

Europe 1987 389 Aplastic anemia (52.7%) MDS (10.5%)

3 Imbert M et al. Europe 1989 213 Malignant myeloid disorders (42%)

Malignant lymphoid disorders (18%)

4 Keisn M et al. Sweden 1990 100 Neoplastic disease (32%)

Aplastic anemia (16%)

5 Hossain M et al. Bangladesh 1992 50 Aplastic anemia Chronic malaria and kalaazar

6 Varma N, Dash S India 1992 202 Aplastic anemia (40.6%)

Megaloblastic anemia (23.26%)

7 Tilak V, Jain R India 1998 77 Megaloblastic anemia (68%)

Aplastic anemia (7.7%)

8 Javage DG et al. Zimbabwe 1999 134 Megaloblastic anemia Aplastic anemia

9 Khodke et al. India 2000 166 Hypoplastic anemia (29.51%)


10 Naeem Khan M et al. Pakistan 2001 30 Aplastic anemia (20%) Megaloblastic

anemia (16.7%)

11 Kumar R et al. India 2001 166 Aplastic anemia (29.5%)


12 Osama Ishtiaq et al. Pakistan 2002 100 Megaloblastic anemia (39%)

Hypersplenism (19%)

13 Mussafrat Niazi et al. Pakistan 2004 89 Aplastic anemia

(38.3%) Megaloblastic anemia (27.7%)

14 Mobina Ahsan Dodhy et al. Pakistan 2005 392 Megaloblastic anemia

(35.95%) Hypersplenism (16.3%)

15 Fazlur Rahim et al. Pakistan 2005 424 Megaloblastic anemia (24.9%)

Aplastic anemia (14.15%)

16 Jhazia Memon et al. Pakistan 2008 230 Aplastic anemia (23.9%)


17 Jha et al. Nepal 2008 148 Hypoplastic anemia (29.5%)


18 Present study India 2010 50 Megaloblastic anemia (44%)

Nutritional anemia (20%)

77

I. MEGALOBLASTIC ANEMIA ASSOCIATED WITH PANCYTOPENIA

Megaloblastic anemia is common in India. This seems to reflect the higher

prevalence of nutritional anemia in Indian subjects.

Age and sex distribution

In the study of pancytopenia cases by Jha et al., the age range was 10-79 years

(31 years). There was a male preponderance and male to female ratio was 1.5:1. In the

study by Kumar et al.70 the ages ranged from 14-73 years (39.5%). There was a

female preponderance and the male to female ratio was 2:1. In the present study,

age ranged from 18-70 years. Majority of the patients were in the age group of

31-50 years (62%). There was a male preponderance and the male to female ratio was

2.4:1.

Hematological Parameters

Jha et al.3 Kumar et al.70 Present study

Hb% (gm/dl) 2.3-9.8 (6.2) 2.4-7 (4.6) 2-10

TLC (cells/mm3) 1200-3900 (2600) 700-3600 (2800) 500-4000

Platelet (C/mm3) 2000-1.37 (50,000) 10,000-1.30 (76,000) 26000-1,50,000

Peripheral Smear

The principal hematologic manifestations are, varying degrees of anemia,

leucopenia, thrombocytopenia, anisopoikilocytosis, macroovalocytosis and hyper-

segmented neutrophils.

In the study by Kishore Khodke et al.2 20/22 cases showed anisocytosis, 10/22

cases showed dimorphic blood picture and 20/22 cases showed hypersegmented

neutrophils. In the study by Tilak et al.10 51/53 cases showed anisocytosis, 45/53

78

cases showed hypersegmented neutrophils, 13/53 showed circulating erythroblasts.

Reticulocytes were seen in 5/53 and relative lymphocytosis was seen in 7/53 cases.

In the present study, macroovalocytes with considerable degree of

anisopoikilocytosis were the main features in all the cases. MCV was more than

100 fl in 57.5% of cases and dimorphic blood picture was seen in 30% of cases (10

patients). Hypersegmented neutrophils were seen in most of the patients.

Bone Marrow

Bone marrow is usually hypercellular with predominantly megaloblastic

erythropoiesis. Giant band forms, metamyelocytes and giant megakaryocytes are also

seen.

In the present study, the bone marrow was hypercellular with reduction of fat

cells in most of the patients (81.8%). Four patients (18.2%) had normocellular

marrow. Erythroid hyperplasia with megaloblastic maturation was seen in all the

patients.

II. NUTRITIONAL ANEMIA (MIXED)

Nutritional anemia as a common etiological factor causing pancytopenia is

well recognised and established. The nutritional deficiency of either B12 or folate

results in megaloblastic anemia. Other causes include mixed deficiency anemia

(microcytes and macrocytic). In the study by Shazia Memon75 mixed deficiency was

seen in 20 cases (8.69%). Mobina et al.4 in their study of 392 cases of pancytopenia

found 11.2% cases of mixed deficiency anemia.

79

In the present study, mixed deficiency was seen in 20% of patients. This

percentage is much lower than expected because 60-80% of world population is

affected by iron deficiency anemia which is the most common preventable nutritional

deficiency in the world. The possible explanation is that, majority of the cases present

with anemia rather than pancytopenia and are diagnosed on smear examination and

treated as outpatients.

The age ranged in the present study from 18-60 years. There was a

male preponderance and male to female ratio was 2.3:1. Most of the patients had

dimorphic anemia. Two patients had macrocytic and hypochromic anemia. Bone

marrow was hypercellular. Erythroid hyperplasia with both megaloblastic and micro

normoblastic maturation was observed in all the patients. Leucopoiesis was normal.

Megakaryopoeisis was either normal or increased.

III. APLASTIC ANEMIA ASSOCIATED WITH PANCYTOPENIA


In the study by Kumar et al.70 the ages ranged from 12-63 years (29 years).

There was a male preponderance and male to female ratio was 1.4:1. In the study by

Jha et al.3 the ages ranged from 1.5-70 years (17 years). There was a male

preponderance with male to female ratio of 1.3:1.

In the present study, the ages ranged from 31-60 years. Majority of the patients

were in the age group of 41-50 years. Aplastic anemia was more common in males.

The male to female ratio was 1.5:1.

80

2. Hematological Parameters

The hematological parameters in aplastic anemia by various studies are

compared with the present study as below.


Hb% (gm/dl) 2.9-5 (5.9) 13-8 (4.4) 3.1-10

TLC (cells/mm3) 900-3800 (2400) 200-3000 (1000) 1100-4000

Platelet (C/mm3) 1000-1.36 (30,000) 8000-86,000 (4500) 5100-1,50,000 3. Peripheral Smears

In the study by Kishore Khodke,2 3/7 patients showed anisocytosis and 1/7

patients showed relative lymphocytosis. In the study by Tilak et al.10 2/6 patients had

anisocytosis and 3/6 patients had relative lymphocytosis. In the study by Daniel

NM et al.15 found normocytic normochromic erythrocytes in 64% of the patients,

macrocytic normochromic blood picture in 20% of the patients.

In the present study, 64.2% had normocytic normochromic erythrocytes.

35.8% of the patients had macrocytic anemia and 56.3% of them had relative

lymphocytosis.

Bone marrow

Cellularity of bone marrow in aplastic anemia is very much reduced. It may be

hypocellular or acellular. Lymphocytes and plasma cells are prominent. Daniel NM in

their analysis of 50 cases reported 74% of patients with hypocellular marrow, 16% of

patients with normocellular marrow which later became hypocellular and 10% with

acellular marrow.

In the present study, bone marrow was mostly hypocellular and the aspirate

was composed of fat cells in all the patients. There was a relative increase in plasma

81

cells and lymphocytes. Bone marrow trephine biopsy revealed replacement of marrow

by fat cells.

IV. HYPERSPLENISM

Hypersplenism is known to cause pancytopenia by sequestration of blood

cells. In a study of 195 patients, Retief HP18 found hypersplenism to be the cause of

pancytopenia in 7.7% of the patients. Shazia Memon et al.75 in their study of 230

cases found hypersplenism in 10 patients (4.34%). Kumar et al.70 reported on

incidence of hypersplenism in 19/166 cases in which ages ranged from 14-49 years.

There was a male preponderance with the male to female ratio being 2:1.

In the present study, hypersplenism was the cause of pancytopenia in 10% of

cases. Ages ranged from 41-70 years. There was a male preponderance with the male

to female ratio being 2:1.

Hematological parameters

In the study by Kumar et al.70 the Hb% ranged from 3.5-8.6 gm%. The TLC

ranged from 1100-3600 cells/cumm. The platelets ranged from 40,000-1,25,000

cells/cumm.

In the present study, the Hb% ranged from 3.8-10 g%, TLC ranged from

1700-3800 cells/cumm and the platelets ranged from 26,000-1,40,000 cells/cumm.

Peripheral smear

Most of the patients (60%) had normocytic normochromic anemia. 40%

of them had microcytic hypochromic anemia. In the study by Osama et al.72

macrocytosis was seen in 63.1% cases and microcytosis in 36.8% cases.

82

Bone marrow

66.7% had hypercellular marrow while the rest had normocellular.

V. LEUKEMIA ASSOCIATED WITH PANCYTOPENIA

Age and sex distribution and incidence

In the study by Jha et al.,3 acute leukemia alone constituted 90.62% of all the

hematological malignancies. It accounted for 19.59% of total cases of pancytopenia.

Age ranged from 2-75 years with a male to female ratio of 1.9:1. Khodke et al.2 and

Tilak et al.10 reported one case of AML causing pancytopenia. Acute leukemia was

the third common cause of pancytopenia in the study of Varma and Dash which is

similar to the study by Savage et al.69

In the present study leukemia accounted for 6% of pancytopenia cases.

Majority (66.7%) of the pancytopenia cases were due to acute leukemia. The ages

varied from 21-40 years. There was a male preponderance with male to female ratio

being 2:1.

Hematological parameters

The hematological parameters in leukemia are compared as below.


Hb% (gm/dl) 1.2-9.6 (5.9) 2.6-6 (4) 5.1-9.3

TLC (cells/mm3) 800-3600 (1950) 650-3200 (2200) 1100-4000

Platelet (C/mm3) 6000-1.25 (37,500) 18000-96000 (52,000) 26000-89000

Peripheral blood picture

In the study by Tilak Jain et al. one case of acute myeloid leukemia with

anisocytosis, circulating erythroblasts and immature cells was reported. Kishore

83

Khodke et al. found one case of acute myeloid leukemia with immature cells in the

peripheral blood.

In the present study, all the patients had normocytic normochromic anemia.

Leukocyte count was reduced and circulating immature cells were seen. Platelet count

was also reduced.

Bone marrow

Bone marrow was hypercellular in all the cases. Myeloid hyperplasia with

immature cells was seen in all the cases. Erythropoiesis and megakaryopoiesis were

reduced.

VI. MYELODYSPLASTIC SYNDROME (MDS)

Pancytopenia is known to occur in MDS. It is the least common finding

encountered in patients with MDS as compared to mono and bicytopenia.

In a study of 816 patients with MDS by Greenberg et al.41 pancytopenia was

found in 15% of the patients. In a study of 31 patients of MDS by Kini J et al.42

bicytopenia was the commonest finding.

Age and sex distribution

In a study of 118 patients with MDS by Juneja SK et al.38 the age ranged from

48-95 years. In a study of 31 patients by Kini J et al.42 the patients were in the age

group of 4-7 years.

In the present study, one case presented with pancytopenia in a female patient

aged 55 years.

84

VII. DENGUE FEVER

Naeem Khan et al.71 studied 30 cases of pancytopenia and found 1 case

of dengue fever. In the present study, 2 cases in 18 years (male) and 22 years

(female) with dengue fever presented with pancytopenia. Total leukocyte count was

800 cells/cumm and platelet count was 28,000 cells/cumm. Bone marrow was

hypercellular showing erythroid hyperplasia with reversal of M:E ratio.

VIII. HEMOLYTIC ANEMIA

Fazlur Rahim et al.76 in their study found three cases of pancytopenia with

hemolytic anemia. Osama et al.72 in their study found two cases of pancytopenia with

hemolytic anemia.

In the present study, one patient of hemolytic anemia presented

with pancytopenia. Peripheral blood showed features of hemolysis. Erythrocytes

showed moderate degree of anisopoikilocytosis and an increase in number of

polychromatophilic RBCs.

Reticulocyte count was markedly increased. Bone marrow was hypercellular

with a reversal of M:E ratio. Erythroid hyperplasia with normoblastic maturation was

seen. Megakaryocytic hyperplasia was noted in both the cases.

85

CONCLUSION

Pancytopenia is a common entity. However, it has received inadequate

attention in the Indian subcontinent. A study of pancytopenia using easily available

diagnostic techniques is therefore important.

Age and sex distribution of patients with pancytopenia in this study was

consistent with the findings in other studies. Megaloblastic anemia was the

commonest cause of pancytopenia in the present study. Most other studies have

reported aplastic anemia as the commonest cause. This seems to reflect higher

prevalence of nutritional anaemia in the Indian subjects. The haematological

parameters and bone marrow morphological features in patients with megaloblastic

anaemia, aplastic anaemia and malignant diseases including MDS in the present study

were comparable to the findings by other authors.

Uncommon etiological factors like dengue fever and hemolytic anemia were

identified in this study.

A comprehensive clinical, haematological and bone marrow study of patients

with pancytopenia usually helps in identification of the underlying cause. However, in

view of a wide array of etiological factors, pancytopenia continues to be a challenge

for hematologists.

86

SUMMARY

Fifty patients with a hematological diagnosis of pancytopenia were studied

during the period November 2008 to May 2010, in the Department of Pathology,

Mysore Medical College and Research Institute, Mysore. The following were the

etiological factors.

1. Megaloblastic anemia – 44%

2. Mixed nutritional anemia – 20%

3. Hypersplenism – 12%

4. Aplastic anemia – 10%

5. Malignant conditions – 6%

6. Myelodysplastic syndromes – 2%

7. Others – 6%

1. Megaloblastic anemia

• This was the most common cause of pancytopenia in the present study.

• There was a male preponderance with male to female ratio of 2.4:1. It was most

common in the age group of 31-50 years.

• Hemoglobin varied from 2 g% to 10g%. The total leukocyte count ranged from

500-4000 cells/cumm. Platelet count ranged from 24000-1.5 lakh cells/cumm.

Reticulocyte count ranged from 0.1% to 2%. MCV was more than 100 fl in 57.5%

of cases.

• Majority of the patients had macrocytic and dimorphic anemia. Hypersegmented

neutrophils were present in all the patients.

87

• Bone marrow was hypercellular. Megaloblastic erythropoiesis with giant meta-

myelocytes and band forms were seen. Megakaryocytes were normal.

2. Mixed nutritional anemia

• This was the second most common cause of pancytopenia in the present study.

• There was a male preponderance with a male to female ratio of 2.3:1. It was most


• Hemoglobin varied from 2.3 g% to 7.8 g%. The total leukocyte count ranged from


Reticulocyte count ranged from 0.1-8%.

• Majority of the patients had normochromic normocytic anemia. Two patients had

microcytic hypochromic anemia.

• Bone marrow was hypercellular with a reversal of M:E ratio in 93.8% of cases.

3. Hypersplenism

• This was the next common cause of pancytopenia in the present study.

• There was a male preponderance with a male to female ratio of 2:1. It was most


• Hemoglobin varied from 3.8 g% to 10 g%. The total leukocyte count ranged from



• Majority of the patients had normochromic normocytic anemia (60%). 40% of

patients had microcytic hypochromic anemia.

• Bone marrow was hypercellular with a reversal of M:E ratio in 70% of cases.

88

4. Aplastic anemia

• This was the next common cause of pancytopenia in the present study.

• There was a male preponderance with a male to female ratio of 1.5:1. It was most


• Hemoglobin varied from 3.1-10 g%. The total leukocyte count ranged from


Reticulocyte count ranged from 0.2%-1.8%.

• Majority of the patients had normochromic normocytic anemia (64.2%). Some

(35.8%) showed macrocytosis. There was relative lymphocytosis.

• Bone marrow was hypocellular with an increase in marrow fat. Lymphocytes and

plasma cells were prominent.

5. Leukemia

• There was a male preponderance with a male to female ratio of 2:1. It was most


• Hemoglobin varied from 5.1-9.8%. The total leukocyte count ranged from

1100-4000 cells/cumm. Platelet count ranged from 76,000-1,00,000 cells/cumm.


• Majority of the patients had normochromic normocytic anemia. Leukocytes were

reduced in number and immature cells including myeloblasts were seen.

• Bone marrow was hypercellular with a reversal of M:E ratio in 80%.

89

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98

PROFORMA

NAME: OP/IP NO:

BM NO:

AGE: D.O.A:

SEX: D.O.D:

UNIT AND WARD:

CHIEF COMPLAINTS: HISTORY OF PRESENTING COMPLAINTS: PAST HISTORY: FAMILY HISTORY: MALIGNANCY / CONGENITAL ABNORMALITIES GENERAL EXAMINATION: SYSTEMIC EXAMINATION:

CVS:

GIT:

RS:

CNS:

EXAMINATION OF LYMPH NODES:

a. SITE: CERVICAL / AXILLARY / INGUINAL

R/L R/L R/L

b. SINGLE / MULTIPLE

c. SIZE / MOBILE / MATTED / TENDERNESS

99

PROVISIONAL DIAGNOSIS:

INVESTIGATIONS:

Hb: TLC:

PLATELET COUNT:

DC:

BT: CT: MCV: MCH:

MCHC:

PCV:

COMPLETE HEMOGRAM: BONE MARROW ASPIRATION / BIOPSY FINDINGS: SPECIAL STAINS USED: IMPRESSION:

100

STAINING TECHNIQUE

Leishman’s Stain

1. Smears were air dried.

2. Slides placed on a slide stand and covered with Leishman’s stain for 5 min.

3. Double the amount of distilled water added over the Leishman’s stain and left for

10 min.

4. Smears washed with distilled water and air dried.

Result: Cytoplasm – Pink; Nucleus – Blue

Myeloperoxidase stain

1. Smears placed in formalin-alcohol fixative for 60 seconds at room temperature.

2. Washed in running tap water for 15-30 seconds.

3. Excess water removed and stain put and left for 30 seconds.

4. Washed in running tap water for 30 to 60 seconds.

5. Slides dried and examined.

(Stain composition: Ethanol, benzidine dihydrochloride, zinc sulphate, sodium

acetate, hydrogen peroxide, sodium hydroxide and safranin)

Results: Nuclei – Red; Peroxidase activity – Blue granules

Periodic acid Schiff stain

1. Fixed air dried smears for 10-15 minutes in formalin-ethanol at room temperature

(10 ml of 40% formalin and 90 ml absolute ethanol).

2. Washed in running tap water for 5-10 minutes.

101

3. Incubated in 1% periodic acid for 20 minutes at room temperature.

4. Washed in running tap water for 5-10 minutes.

5. Incubated in Schiff reagent at room temperature.

6. Washed in running tap water for 10 minutes.

7. Counter stained with Harris’ hematoxylin for 5-10 minutes.

8. Washed in running tap water for 5 minutes and air dried.

Results: Nuclei – Blue. Substances which show positive reaction: Magenta Pink.

102

STATISTICAL METHODS APPLIED

Frequencies

The Frequencies procedure provides statistics and graphical displays that are

useful for describing many types of variables. For a first look at your data, the

Frequencies procedure is a good place to start.

For a frequency report and bar chart, you can arrange the distinct values in

ascending or descending order or order the categories by their frequencies. The

frequencies report can be suppressed when a variable has many distinct values. You

can label charts with frequencies (the default) or percentages.

Descriptives

The Descriptives procedure displays univariate summary statistics for several

variables in a single table and calculates standardized values (z scores). Variables can

be ordered by the size of their means (in ascending or descending order),

alphabetically, or by the order in which you select the variables (the default).

Crosstabs

The Crosstabs procedure forms two-way and multiway tables and provides a

variety of tests and measures of association for two-way tables. The structure of the

table and whether categories are ordered determine what test or measure to use.

Chi-Square Test

The Chi-Square Test procedure tabulates a variable into categories and

computes a chi-square statistic. This goodness-of-fit test compares the observed and

expected frequencies in each category to test either that all categories contain the

same proportion of values or that each category contains a user-specified proportion

of values.

All the statistical methods were carried out through the SPSS for Windows (version 16.0)

103

KEY TO MASTER CHART

AA Aplastic anemia

AML Acute myeloid leukemia

F Female

HA Hemolytic anemia

HS Hypersplenism

M Male

MA Megaloblastic anemia

MDS Myelodysplastic syndrome

N Normal

NA Nutritional anemia

NC Normocellular

↑ Hypercellular

↓ Hypocellular

Sl. No. Name Age

(years) Sex BM No. OP No. IP No. Chief complaint Salient exam findings Hb% TC PLT PS BM

cellularity1 Anwar Pasha 28 M 27\08 22734 Generalised weakness Pallor 2.2 1000 50,000 Pancytopenia ↑

2 Revathi 22 F 28\08 25921 Generalised weakness Spleen+ 2.8 1500 40,000 Pancytopenia ↑

3 Noor Ahmed 25 M 1\09 25258 Generalised weakness Pallor 4.1 2000 55,000 Pancytopenia ↑

4 Sannamadaiah 48 M 2\09 731/08 Abdominal distension Spleen++ 3.5 2000 60,000 Pancytopenia ↑

5 Ranjitha 21 F 3\09 30022 Fever Pallor spleen+ 3 3000 1,00,000 Pancytopenia ↑

6 Siddappa 55 M 4\09 31052 Generalised weakness Pallor 4.7 2000 70,000 Pancytopenia NC

7 Parvathamma 42 F 5\09 1602 Fever Pallor 6.5 1500 55,000 Pancytopenia ↓

8 Lakshmi 33 F 6\09 734/08 Generalised weakness Pallor 6 2100 75,000 Pancytopenia ↑

9 Chikkanna 38 M 7\09 8722 Generalised weakness Pallor 3.7 2000 75,000 Pancytopenia ↓

10 Kumar 26 M 8\09 7730 Generalised weakness Pallor 2 1000 25,000 Pancytopenia NC

11 Mohan 27 M 9\09 10928 Fever Pallor 5.5 2000 50,000 Pancytopenia ↑

12 Raniamma 37 F 10\09 37621 Generalised weakness Pallor 5.2 2500 60,000 Pancytopenia NC

13 Raja 20 M 11\09 37769 Generalised weakness Pallor 3.5 2500 25,000 Pancytopenia ↑

14 Karigowda 53 M 12\09 38861 Generalised weakness Pallor 4 3000 50,000 Pancytopenia ↑

15 Parvathi 53 F 13\09 38872 Fever Pallor 3.9 3000 1,00,000 Pancytopenia ↓

16 Manjunatha 32 M 14\09 790/08 Generalised weakness Pallor 5.2 3000 65,000 Pancytopenia ↑

17 Mahadeva 34 M 15\09 38970 Generalised weakness Pallor 7 2500 60,000 Pancytopenia ↑

18 Mahadevamma 45 F 16\09 39900 Generalised weakness Pallor 4 2200 50,000 Pancytopenia ↑

19 Pushpa 48 F 17\09 39912 Abdominal distension Spleen++ 5.5 1500 75,000 Pancytopenia ↑

20 Raju 38 M 18\09 39970 Generalised weakness Pallor 6.5 2200 1,00,000 Pancytopenia NC

21 Murali 33 M 19\09 40120 Generalised weakness Pallor 6 2500 1,00,000 Pancytopenia ↑

22 Shanthi 60 F 20\09 40170 Generalised weakness Pallor 6.8 3000 90,000 Pancytopenia ↑

23 Krishnamurthy 37 M 21\09 41181 Generalised weakness Pallor 7 2200 76,000 Pancytopenia NC

24 Puttanna 57 M 22\09 74/09 Generalised weakness Pallor 3.5 4000 40,000 Pancytopenia ↑

25 Jyothi 58 F 23\09 41190 Generalised weakness Pallor 6.5 3000 70,000 Pancytopenia ↑

104

Sl. No. Name Age

(years) Sex BM No. OP No. IP No. Chief complaint Salient exam findings Hb% TC PLT PS BM

cellularity26 Mahadevappa 58 M 24\09 41200 Generalised weakness Pallor 6 2500 55,000 Pancytopenia ↑

27 Surendra 18 M 25\09 41290 Fever Pallor,Bleeding 5 800 28,000 Pancytopenia ↑

28 Venkatesh 41 M 26\09 42321 Generalised weakness Pallor 5.8 4000 80,000 Pancytopenia ↑

29 Somanna 45 M 27\09 42400 Generalised weakness Pallor 5.5 3500 1,20,000 Pancytopenia ↑

30 Puttamma 58 F 28\09 42471 Pain abdomen Pallor 4 3500 75,000 Pancytopenia ↑

31 Prabhu 43 M 29\09 42521 Pedal edema Pallor 5.2 3200 1,00,000 Pancytopenia ↑

32 Narendra 48 M 30\09 120/09 Generalised weakness Pallor 7 3700 75,000 Pancytopenia ↑

33 Sarojamma 56 F 1\10 42602 Abdominal distension Spleen++ 7.5 2000 50,000 Pancytopenia ↑

34 Kalidasa 45 M 2\10 42630 Fever Pallor 5.5 3200 75,000 Pancytopenia ↑

35 Abdul 43 M 3\10 150/60 Joint pain Pallor 6.8 4000 80,000 Pancytopenia ↓

36 Raju 42 M 4\10 42732 Generalised weakness Pallor 7.4 4000 1,50,000 Pancytopenia ↓

37 Puttathayamma 55 F 5\10 43100 Abdominal distension Pallor,Spleen++ 4 3000 1,00,000 Pancytopenia ↑

38 Shekar 59 M 6\10 43232 Generalised weakness Pallor 6 4000 60,000 Pancytopenia ↑

39 Jaleel 33 M 7\10 43702 Generalised weakness Pallor 6.5 3500 75,000 Pancytopenia ↑

40 Boregowda 62 M 8\10 43813 Abdominal distension Pallor,Spleen++ 8 4000 75,000 Pancytopenia ↑

41 Anand 44 M 9\10 43840 Generalised weakness Pallor 6.5 4000 1,00,000 Pancytopenia ↑

42 Puttegowda 66 M 10\10 232/10 Generalised weakness Pallor 9.6 3800 1,50,000 Pancytopenia ↑

43 Priya 18 F 11\10 238/10 Fever Pallor 7.5 3600 1,50,000 Pancytopenia ↑

44 Wasim Khan 47 M 12\10 43900 Generalised weakness Pallor 7.5 3500 1,50,000 Pancytopenia ↑

45 Srinivas 55 M 13\10 43942 Abdominal distension Spleen++ 7.5 3500 1,00,000 Pancytopenia NC

46 Deepak 39 M 14\10 48212 Fever Pallor 7.6 3500 1,50,000 Pancytopenia ↑

47 Mahalakshmi 32 F 15\10 48318 Fever Gum bleed 8 4000 75,000 Pancytopenia ↑

48 Vijayakumar 58 M 16\10 340/10 Abdominal distension Spleen++ 6 3400 1,50,000 Pancytopenia NC

49 Manjunath M 49 M 17\10 348/10 Generalised weakness Pallor 8 3500 1,20,000 Pancytopenia ↑

50 Geetha 22 F 18\10 49120 Fever Pallor 5 800 30,000 Pancytopenia ↑

106

Sl. No. Erythropoiesis Myelopoiesis Megakaryopoiesis BM diagnosis Final

impression1 Hyperplasia and megaloblastic Megaloblastic N MA MA

2 Hyperplasia and megaloblastic N N MA MA

3 Megaloblastic Megaloblastic N MA MA

4 Hyperplasia N N Erythroid hyperplasia with normoblastic maturation HS

5 Hyperplasia N N Erythroid hyperplasia with normoblastic maturation HA


7 Hyperplasia Hypoplasia Hypoplasia AA AA

8 Megaloblastic Megaloblastic Megaloblastic MA MA

9 Hypoplasia Hypoplasia Hypoplasia AA AA

10 Hyperplasia N N Erythroid hyperplasia with normoblastic maturation NA

11 Decreased Hyperplasia and megaloblastic Decreased AML AML

12 Megaloblastic N N MA MA

13 Hyperplasia and normoblastic N N Erythroid hyperplasia with normoblastic maturation NA

14 Hyperplasia N N Erythroid hyperplasia with micronormoblastic maturation NA

15 Hyperplasia Hyperplasia Hyperplasia AA AA



18 Hyperplasia and normoblastic N N Erythroid hyperplasia with normoblastic maturation NA








105

Sl. No. Erythropoiesis Myelopoiesis Megakaryopoiesis BM diagnosis Final

impression26 Megaloblastic N N MA MA

27 Hyperplasia N N Reactive erythroid hyperplasia Dengue




31 Megaloblastic Megaloblastic Megaloblastic MA MA






37 Megaloblastic Dysmyelopoiesis Dysmegakaryopoiesis MDS MDS



40 Hyperplasia N N Reactive erythroid hyperplasia HS





45 Hyperplasia N N Erythroid hyperplasia with micronormoblastic maturation HS

46 Decreased Hyperplasia,blasts+ Decreased AML AML

47 Decreased Increased myeloblasts Decreased AML AML



50 Hyperplasia Hyperplasia N Reactive erythroid hyperplasia Dengue

107

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