ANALYSIS OF PERIPHERAL SMEAR AND BONE MARROW

MORPHOLOGY IN EVALUATION OF PANCYTOPENIA /

BICYTOPENIA – A PROSPECTIVE STUDY

Dissertation submitted in

Partial fulfillment of the regulations required for the award of

M.D. Degree

in

PATHOLOGY - BRANCH III

THE TAMILNADU

DR.M.G.R.MEDICAL UNIVERSITY

CHENNAI

MAY 2019

DECLARATION

I hereby declare that the dissertation entitled “ANALYSIS OF

PERIPHERAL SMEAR AND BONE MARROW MORPHOLOGY

IN EVALUATION OF PANCYTOPENIA/BICYTOPENIA -

A PROSPECTIVE STUDY ” is a bonafide research work done by me in the

Department of Pathology, Coimbatore Medical College during the period from

NOVEMBER 2016 TO JUNE 2018 under the guidance and supervision of

Dr. VINUTA MALAICHAMY, M.D, Senior Assistant Professor, Department

of Pathology, Coimbatore Medical College.

This dissertation is submitted to The Tamilnadu Dr.M.G.R Medical

University, Chennai towards the partial fulfillment of the requirement for the

award of M.D., Degree ( Branch III ) in Pathology. I have not submitted this

dissertation on any previous occasion to any University for the award of any

Degree.

Place : Coimbatore Dr. B.SOPHIYA

Date :

CERTIFICATE

This is to certify that dissertation entitled “ ANALYSIS OF

PERIPHERAL SMEAR AND BONE MARROW MORPHOLOGY

IN EVALUATION OF PANCYTOPENIA/BICYTOPENIA -

A PROSPECTIVE STUDY ” is a bonafide work done by Dr. B. SOPHIYA,

a postgraduate student in the Department of Pathology, Coimbatore Medical

College, Coimbatore under guidance and supervision of Dr. VINUTA

MALAICHAMY, M.D., Senior Assistant Professor, Department of Pathology,

Coimbatore Medical College, Coimbatore in partial fulfillment of the

regulations of the Tamil Nadu Dr. M.G.R. Medical University, Chennai towards

the award of M.D. Degree (Branch III) in Pathology.

Guide Head of the Department

Dr. Vinuta Malaichamy, M.D., Dr. C.Lalitha, M.D.,

Senior Assistant Professor, Professor,

Department of Pathology, Department of Pathology,

Coimbatore Medical College, Coimbatore Medical College,

Coimbatore. Coimbatore.

Dr. B.Asokan, M.S., M.Ch.,

Dean,

Coimbatore Medical College,

Coimbatore.

ACKNOWLEDGEMENT

To begin with, I thank the almighty God for bestowing his blessing on me

in this dissertation a successful one.

I wish to thank our beloved Dean Dr.B.ASOKAN. M.S., M.Ch ,

Coimbatore Medical College and Hospital, Coimbatore for permitting me to

conduct this study.

I thank Dr. C.Lalitha. M.D., Professor and Head of the Department,

Department of Pathology, Coimbatore Medical College, Coimbatore for her

guidance and support.

I express my gratitude and sincere thanks to my guide Dr. Vinuta

Malaichamy, M.D., Senior Assistant Professor, Department of Pathology,

Coimbatore Medical College, Coimbatore. This dissertation bears her valuable

suggestions and unparalleled professional advice.

I thank my Associate Professors and Assistant Professors of Department

of Pathology, Coimbatore Medical College, Coimbatore for their opinion and

encouragement.

I thank Department of Medicine, Coimbatore Medical College,

Coimbatore for providing clinical cases, valuable support and guidance which

made this dissertation possible.

My deepest and most heart whelming thanks goes to my parents. My

father V. Balasundaram and my mother L.Manonmani have showered me

with lots and lots of love.

I thank my husband Dr. M.S.Kandasamy Kamindan, M.S., whose love,

support and constant patience have taught me so much about sacrifice,

discipline and compromise.

I have nothing but love and appreciation to my daughter Poovithal

Kamindan for understanding and being patient during the time i took for the

study. Her smile keeps me going.

I thank all my non-teaching staffs working in Department of Pathology,

Coimbatore Medical College, Coimbatore.

Dr. B.SOPHIYA

CERTIFICATE - II

This is to certify that this dissertation work titled ANALYSIS OF

PERIPHERAL SMEAR AND BONE MARROW MORPHOLOGY

IN EVALUATION OF PANCYTOPENIA / BICYTOPENIA - A

PROSPECTIVE STUDY of the candidate Dr. B.SOPHIYA with registration

number 201613256 for the award of M.D Degree in the branch of

PATHOLOGY. I personally verified the urkund.com website for the purpose

of plagiarism check. I found that the uploaded thesis file contains introduction

to conclusion pages and result shows 6% percentage of plagiarism in the

dissertation.

Guide & Supervisor

CONTENTS

S.NO PARTICULARS PAGE NO.

1. INTRODUCTION 1-2

2. AIMS AND OBJECTIVES 3

3. NORMAL HEMATOPOIESIS 4-14

4. REVIEW OF LITERATURE 15-39

5. MATERIALS AND METHODS 40-51

6. OBSERVATION AND RESULTS 52-69

7. DISCUSSION 70-79

8. CONCLUSION 80

9. BIBLIOGRAPHY 81

10. ANNEXURES

I: MASTER CHART

II: ABBREVIATIONS

III: PROFORMA

LIST OF TABLES

S.

NO

TITLE PAGE

NO.

1. MEAN AGE OF THE STUDY 52

2. AGE DISTRIBUTION IN THE STUDY 52

3. GENDER DISTRIBUTION IN THE STUDY 53

4. MEAN WHITE BLOOD CELL COUNT OF THE STUDY 55

5. MEAN HEMOGLOBIN VALUE OF THE STUDY 55

6. MEAN PLATELET COUNT OF THE STUDY 55

7. MEAN MCV VALUE OF THE STUDY 55

8. MEAN WHITE BLOOD CELL COUNT IN

PANCYTOPENIA VERSUS BICYTOPENIA

57

9. MEAN HEMOGLOBIN VALUE IN PANCYTOPENIA

VERSUS BICYTOPENIA

58

10. MEAN PLATELET COUNT IN PANCYTOPENIA VERSUS

BICYTOPENIA

59

11. MEAN MCV VALUE IN PANCYTOPENIA VERSUS

BICYTOPENIA

60

12. PERIPHERAL SMEAR DIAGNOSIS 61

13. TYPE OF ANEMIA IN PERIPHERAL SMEAR 62

14. TYPE OF ANEMIA IN PANCYTOPENIA VERSUS

BICYTOPENIA

64

15. BONE MARROW DIAGNOSIS 66

16. BONE MARROW DIAGNOSIS IN PANCYTOPENIA

VERSUS BICYTOPENIA

68

17. COMPARISON OF VARIOUS ETIOLOGY OF

PANCYTOPENIA/ BICYTOPENIA IN DIFFERENT

STUDIES

73

LIST OF CHARTS

S.

NO. TITLE

PAGE

NO.

1. AGE DISTRIBUTION IN THE STUDY 53

2. GENDER DISTRIBUTION IN THE STUDY 54

3. MEAN LABORATORY VALUES 56

4. MEAN WHITE BLOOD CELL COUNT IN

PANCYTOPENIA VERSUS BICYTOPENIA

57

5. MEAN HEMOGLOBIN VALUE IN PANCYTOPENIA

VERSUS BICYTOPENIA

58

6. MEAN PLATELET COUNT IN PANCYTOPENIA

VERSUS BICYTOPENIA

59

7. MEAN MCV VALUE IN PANCYTOPENIA VERSUS

BICYTOPENIA

60

8. PERIPHERAL SMEAR DIAGNOSIS 61

9. TYPE OF ANEMIA IN PERIPHERAL SMEAR 63

10. TYPE OF ANEMIA IN PANCYTOPENIA VERSUS

BICYTOPENIA

65

11. BONE MARROW DIAGNOSIS 67

12. BONE MARROW DIAGNOSIS IN PANCYTOPENIA

VERSUS BICYTOPENIA

69

LIST OF COLOUR PLATES

S.NO TITLE

1 MACROCYTIC ANEMIA IN LEISHMAN STAINED

PERIPHERAL SMEAR

2 HYPERSEGMENTED NEUTROPHIL IN LEISHMAN STAINED

PERIPHERAL SMEAR

3 MEGALOBLASTIC ANEMIA IN BONE MARROW ASPIRATE

4 DIMORPHIC ANEMIA IN LEISHMAN STAINED PERIPHERAL

SMEAR

5 DIMORPHIC ANEMIA IN LEISHMAN STAINED BONE

MARROW ASPIRATE

6 MICROCYTIC HYPOCHROMIC ANEMIA IN LEISHMAN

STAINED PERIPHERAL SMEAR

7 MICRONORMOBLASTIC MATURATION IN LEISHMAN

STAINED BONE MARROW ASPIRATE

8 GIANT PLATELET IN LEISHMAN STAINED PERIPHERAL

SMEAR IN IMMUNE THROMBOCYTOPENIA

9 HYPOLOBATED MEGAKARYOCYTE IN LEISHMAN STAINED

BONE MARROW ASPIRATE OF IMMUNE

THROMBOCYTOPENIA

10 DYSPLASTIC ERYTHROID LINEAGE IN LEISHMAN STAINED

BONE MARROW ASPIRATE OF MYELODYSPLASTIC

SYNDROME (MDS)

11 DYSPLASTIC NEUTROPHILS IN LEISHMAN STAINED BONE

MARROW ASPIRATE OF MDS

12 DYSPLASTIC MEGAKARYOCYTES IN LEISHMAN STAINED

BONE MARROW ASPIRATE OF MDS

13 ACUTE LYMPHOBLASTIC LEUKEMIA IN LEISHMAN

STAINED BONE MARROW ASPIRATE

14 ACUTE MYELOID LEUKEMIA IN LEISHMAN STAINED

PERIPHERAL SMEAR

15 ACUTE MYELOID LEUKEMIA IN LEISHMAN STAINED BONE

MARROW ASPIRATE

16 SUDAN BLACK B STAIN OF ACUTE MYELOID LEUKEMIA

17 SUBLEUKEMIC LEUKEMIA IN LEISHMAN STAINED BUFFY

COAT SLIDE

18 METASTATIC DEPOSITS IN LEISHMAN STAINED BONE

MARROW ASPIRATE

19 ROULEAUX FORMATION IN LEISHMAN STAINED

PERIPHERAL SMEAR

20 PLASMA CELLS IN LEISHMAN STAINED BONE MARROW

ASPIRATE OF PLASMACYTOMA

INTRODUCTION

1

INTRODUCTION

Sir William Harvey described blood as the fountain of life and the

primary seat of the soul. The marrow of our bones is the seedbed of our blood¹.

In our day to day practice, Pancytopenia and bicytopenia is an important

clinico-hematological disorder. Pancytopenia is defined as reduction in all the

three cellular elements of blood, that is, red cells, white cells and platelets.

Bicytopenia is reduction in any of the two cell lines².

It is not a disease entity, but a triad of findings that may result from

various disease processes primarily or secondarily involving the bone marrow.

The etiology of pancytopenia and bicytopenia ranges from transient marrow

viral suppression to marrow infiltration by malignant process. Also caused

iatrogenically secondary to radiotherapy, chemotherapy and drugs³.

A careful examination of the blood film is often helpful in giving a lead

to diagnosis and bone marrow examination usually establishes the diagnosis4. In

cytopenias, the cellularity of bone marrow and its composition varies with the

causes. The bone marrow is normocellular or hypercellular in case of cytopenias

resulting from ineffective hematopoiesis, bone marrow infiltration and increased

peripheral utilization or destruction of cells. The marrow is hypocellular in

cases of primary production defects.

2

The presenting symptoms are usually related to anemia,

thrombocytopenia and rarely leucopenia. The common clinical manifestations

are pallor, fatigue, splenomegaly, lymphadenopathy, fever, bleeding, weight

loss, hepatomegaly and jaundice5.

The essential investigations required for a diagnosis of pancytopenia and

bicytopenia are hematology and bone marrow examination. Other tests include

radiological, microbiological and biochemical investigations in selected case6.

The management and prognosis of the patients depends on the severity of

cytopenias and the underlying pathology. The present study has been

undertaken to evaluate the various causes and to correlate the peripheral blood

findings with bone marrow aspirates. The data would help in planning the

diagnostic and therapeutic approach in patients with pancytopenia and

bicytopenia.

AIMS AND OBJECTIVES

3

AIMS AND OBJECTIVES

1. To study the various causes of pancytopenia / bicytopenia at Coimbatore

Medical College Hospital,Coimbatore.

2. To correlate and analyse the hematological indices, peripheral smear and

bone marrow morphology in etiological diagnosis of pancytopenia /

bicytopenia patients.

NORMAL HEMATOPOIESIS

4

NORMAL HEMATOPOIESIS

Hemopoietic stem cells are the backbone of the adult blood system, as

they help sustain all blood cells throughout life. These cells, which form the

basis of the blood system are characterized by their ability to self-renew. They

do so by a process of asymmetric cell division, which produces an identical

HSC and a differentiating cell. The daily requirement of erythrocytes and

leukocytes needed to maintain life is provided by the HSCs7.

There are various haemopoietic sites in adult, like spleen, thymus and

lymph nodes. They help in differentiation of a particular haemopoietic cell

lineages and subsets along with supporting them in their growth. These sites

play a key role in the haemopoietic system and playing an equally important and

vital role is blood itself. It functions as transport medium for the circulation of

the mature blood cells. HSCs at low frequency also use blood as transport

system to reach these sites from the marrow8.

At 21 days of gestation appears the very first hemopoiesis in the blood

islands of an extra embryonic yolk sac. At about 28 to 40 days HSCs can be

seen appearing from the aorta – gonad – mesonephros region, within the ventral

wall of dorsal aorta. Apart from these regions, they can also be found in the

vitelline and umbilical arteries and the placenta. These cells arise in

approximation to the endothelial cells9.

5

At 30 – 40 days, fetal liver starts producing erythroid cells and takes

another 20 days for them to be released into the circulation. By the end of 10-12

weeks occurs the migration of the hemopoiesis to bone marrow and by the last 3

months of a fetal life the system is well-developed and established in the

marrow¹º.

Epigenetic patterns like changes in nuclear position, replication timing,

chromatin modification, DNA methylation and internal transcriptional programs

play a complex role in the cells committing to one particular lineage. They may

also receive external signals from the microenvironment (e.g. cytokines, growth

factors and cell – cell interactions) acting via signal transduction pathways¹¹.

The multipotent (or pluripotent) HSC undergo gradual restriction in their

hemopoietic potential as they eventually give rise to unipotent progenitor cells.

The progeny of HSC therefore become progressively restricted to one cell

lineage and they lose the capacity to self-renew. The committed progenitor cells

are branched into:

1. Common myeloid progenitor (CMP). It gives rise to cells of all myeloid

lineages (i.e. granulocytic, erythroid, megakaryocytic). The CMP subsequently

generates granulocyte-macrophage progenitors (GMP)and megakaryocyte-

erythroid progenitors (MEP).

2. Common lymphoid progenitor (CLP). It gives rise to T- and B-lymphocytes

and natural killer (NK) cells.

6

THE ADULT HEMOPOIETIC HIERARCHY

7

ERYTHROPOIESIS

Erythropoietin(EPO), the glycoprotein hormone is a key factor in

determining the rate of red cell production. It is produced mainly by the

peritubular cells of the Kidney in adults. EPO receptors are expressed on

erythroid progenitor cells and the binding of EPO to its receptor results in the

activation of JAK2 tyrosine kinase. It causes tyrosine phosphorylation in a

number of proteins and triggers the activation of several signal transduction

pathways involved in proliferation and in the prevention of apoptosis.

An important effect of EPO is, therefore, to maintain the viability and

proliferation of erythroid progenitor cells, by preventing apoptosis. In synergy

with stem cell factor (SCF), granulocyte-monocyte colony stimulating factor

(GM-CSF) , interleukin (IL)-3 and insulin-like growth factor-1 (IGF-1), EPO

stimulates the rate of differentiation of CFU-E to pronormoblasts. EPO also

stimulates terminal differentiation and decreases the time taken for the

maturation of a pronormoblast to a marrow reticulocyte and its release into the

circulation. The plasma level of EPO is inversely related to the capacity of the

blood to deliver oxygen to the kidneys and other tissues. Reduction of the

oxygen supply to the kidney results in enhanced EPO gene expression via a

hypoxia-regulated transcription factor, HIF (hypoxia inducible factor)12-15

.

8

In Romanowsky-stained normal bone marrow smears erythroid cells

have the following features:

1. Pronormoblasts : It's a large cell (diameter 12–20μm) with a large rounded

nucleus, surrounded by deeply basophilic cytoplasm whose intensity of

basophilia is greater than that of myeloblasts. Cytoplasm adjacent to the nucleus

stains pale corresponds to Golgi Apparatus. Nuclear chromatin is finely granular

or reticular and the nucleus contains a prominent nucleoli.

2. Basophilic normoblasts : Cytoplasm is more blue-staining than

pronormoblast. Nuclear chromatin is more granular and lacks nucleoli.

3. Early polychromatic normoblasts : The cytoplasm is polychromatic due to

hemoglobinization. The nucleus contains condensed chromatin in clumps.

4. Late polychromatic normoblasts : These are smaller cells (diameter 8–

10μm), and have faintly polychromatic cytoplasm and a small eccentric nucleus

(diameter less than 6.5μm), which contains large clumps of condensed

chromatin. The nucleus becomes pyknotic, is extruded and rapidly

phagocytosed by adjacent macrophages16-18

.

5. Reticulocyte : They are rounded anucleate cells that are about 20% larger in

volume than mature red blood cells. It appears polychromatic and mature into

red blood cell over a period of 1-2 days.

9

6. Red blood cells : They are circular biconcave discs with a mean diameter of

7.2μm. It appears red with central pallor.

GRANULOPOIESIS

Granulopoiesis is the process of production of granulocytic cells within

the bone marrow. The characteristic feature of the cells is the presence of

cytoplasmic granules, include Neutrophil, Eosinophils, Basophils and cells of

monocyte-macrophage series. Differentiation of the HSC to common myeloid

progenitor (CMP) marks the initiation of the granulopoiesis. Next step is the

development of bipotent granulocyte-macrophage progenitor (GMP) from the

CMP. Further maturation leads to formation of cells that irreversibly mature to

granulocytic cells (CFU-G) or macrophages (CFU-M)19-21

.

The G-CSF, GM-CSF stimulates the formation of neutrophil

granulocytes from the GMP and is also influenced by M-CSF, SCF and IL-6.

The differentiation of myeloblast follows a sequence. It starts from myeloblast

and differentiate to promyelocyte, myelocyte, metamyelocyte, band form and

finally into the mature segmented neutrophil granulocytes. Cell division occurs

only till the myelocyte stage²²,²³. Cell differentiation occur in both proliferating

and non-proliferating cells. The whole process takes about 10 – 12 days and by

the end of 12 days the mature granulocytes enters the bloodstream. Average T ½

of neutrophils in a healthy individual is 7.2 hours.

10

1. Myeloblasts (10-20μm) – Round cells, contains large round/oval nucleus and

small amount of basophilic agranular cytoplasm with immature nuclear

chromatin and may have two or more nucleoli.

2. The neutrophil promyelocyte – Slightly larger than myeloblasts. It has

ovoid nucleus which is eccentrically placed with a coarser nuclear chromatin

and has a prominent nucleoli. Cytoplasm contains few or more azurophilic

granules(primary granules) with retaining some amount of basophilia.

3.The neutrophil myelocyte – Smaller than promyelocyte and contains more

acidiophilic cytoplasm. The cytoplasm has neutrophilic granules(specific

granules) in addition to the primary azurophilic granules. The nucleus is

rounded, oval, flattened on one side or slightly indented and contain coarsely

granular chromatin and usually lack nucleolus24-26

.

4. Neutrophil metamyelocytes – These cells are smaller than myelocytes. The

nucleus is C shaped and greater chromatin condensation than a myelocyte

nucleus. In the acidophilic cytoplasm contains more neutrophilic granules

leaving only few azurophilic granules27,28

.

5. The band form – The nucleus is narrow band like and usually in the shape of

U, with same chromatin condensation as that of metamyelocytes. The band like

nucleus may be twisted with one or more constrictions29,30

.

11

6. The neutrophil granulocyte – Its slightly smaller and has segmented

nucleus which contain 2-5 nuclear lobes connected with each other by fine

strands of chromatin. If the length of a constriction is more than a half or two-

thirds the breadth of the nucleus, it is considered as adequate evidence of

lobulation³¹,³².

A SEMI-DIAGRAMMATIC REPRESENTATION OF

GRANULOPOIESIS AND ERYTHROPOIESIS

12

MEGAKARYOPOIESIS

The process of development of megakaryocytes and platelets within the

marrow is described as Megakaryopoiesis. Human marrow has the ability to

make 10¹¹ platelets per day and has the potential to increase it to 20-fold if

needed. A cascade of differentiation from the megakaryocyte erythroid

progenitor (MEP) results in megakaryocytes. The commitment of MEP to

megakaryopoiesis is influenced by the thrombopoietin (primary regulator of

platelet production), IL-6 and IL-11, which then forms the Megakaryocyte

colony forming units (CFU-MK).

The CFU-MK, a diploid cell population in which the DNA synthesis

and nuclear division (karyokinesis) is followed by cell division(cytokinesis).

After further maturation the CFU-MK becomes the megakaryoblasts, which are

the earliest recognizable member of megakaryocyte33-36

. The unique feature of

thrombocytopoiesis is endomitosis. This refers to nuclear division with

cytoplasmic maturation but without cell division. As the cell matures from

megakaryoblasts to the megakaryocyte, there is gradual increase in cell size,

number of nuclear lobes and gradual decrease in cytoplasmic basophilia37-40

.

13

Four types of megakaryocytic cells can be identified in Romanowsky-

stained bone marrow smears. These are, in increasing Order of maturity:

1. Megakaryoblasts (group I megakaryocytes) (20–30μm diameter)- have a

single large oval, kidney-shaped or lobed nucleus with several nucleoli. It has a

very high nucleus to cytoplasm ratio and deeply basophilic agranular cytoplasm.

2. Promegakaryocytes (group II megakaryocytes)- larger than megakaryoblast

with low nucleus to cytoplasm ratio and overlapping nuclear lobes. The

cytoplasm is less basophilic and may contain azurophilic granules.

3.Granular megakaryocytes (group III megakaryocytes),which produce

platelets (70 µm in diameter) - possess abundant pale-staining cytoplasm and

numerous azurophilic cytoplasmic granules. The nucleus has coarsely granular

chromatin and multiple lobes.

4. Bare nuclei - it follows after completion of platelet production.

Megakaryocyte has a network of highly specialized membranes called

as the Demarcation membrane system (DMS), dense bodies, secretory vesicles

and other organelles41

. DMS produces long extensions, which undergo

evagination to form the pro platelet processes. These cytoplasmic processes then

fragment to produce platelets of 1-3μm42

. After the fragmentation, almost Bare

nucleus that remains after release is surrounded by nothing but a thin rim of

cytoplasm containing few granules and organelles43

.

14

Each megakaryocyte has the potential to generate 1000-3000 platelets.

In approximately 6 days a megakaryoblast matures into a platelet producing

granular megakaryocyte44-46

. Majority of these cells are in the marrow, but few

cells may escape the marrow through the sinusoids. Such cells commonly get

trapped in the lungs and continue to produce platelets47

.

REVIEW OF LITERATURE

15

REVIEW OF LITERATURE

ETIOLOGY

Pancytopenia with hypocellular bone marrow¹

1. Acquired aplastic anemia

2. Inherited aplastic anemia

-Fanconi anemia

-Dyskeratosis congenita

-Shwachman - Diamond syndrome

-Amegakaryocytic thrombocytopenia

-Reticular dysgenesis

3. Hypoplastic myelodysplastic syndrome

4. Large granular lymphocytic leukemia

5. Hypoplastic PNH

Pancytopenia with hypercellular bone marrow4

A. Primary marrow disorders

1. Acute leukemia

2. Lymphomas

3. Hairy cell leukemia

4. Myelofibrosis

5. Myelodysplastic syndrome

6. Paroxysmal nocturnal hemoglobinuria

16

7. Multiple myeloma

8. Bone marrow metastasis

B. Systemic disorders

1. Hypersplenism

2. Deficiency of vitamin B12 and folic acid

3. Infections such as tuberculosis, kala-azar, brucellosis

4. Alcohol

5. Autoimmune disorders like systemic lupus erythematosus

PATHOGENESIS

Primary defect in aplastic anemia is in the haemopoietic stem cell itself

or is the result of an environmental factors, particularly immunological attack

on the cell. It is likely that antigens derived from infections with a virus,

exposure to drugs or chemicals or neoantigens from a somatic genetic event are

processed by the immune system and leads to T-cell activation against the

specific offending antigen and probably more importantly, also against normal

cellular antigens which are excessively/aberrantly expressed in the

hematopoietic cells. Central to these late events in the immunosuppression of

hematopoiesis are the cytokines interferon -gamma, interleukin-2 and tumor

necrosis factor, which are secreted by activated immune system. These also

induce expression of the Fas receptor on CD34+ hematopoietic progenitor cells,

leading to their apoptotic cell death. Telomere shortening also seen in some

patients48,49

.

17

Myelodysplastic syndrome is a clonal disorder which originates in a

pluripotent hematopoietic stem cell. The initial event is a somatic mutation at

the level of stem cell that results in the formation of functionally and

structurally defective blood cells having shortened survival. Increased blood cell

proliferation in marrow together with enhanced apoptosis lead to ineffective

erythropoiesis, formation of defective cells that are rapidly removed from the

circulation causing peripheral cytopenia. The defective clone has growth

advantage over the normal hematopoietic cells so that it expands gradually and

suppresses normal hematopoiesis. The phenotypic expression of the pathologic

clone is variable and can manifest as abnormalities of erythrocytic,

granulocytic, monocytic or megakaryocytic cell lines. The pathological clone is

unstable, new genetic insults can superimpose on the original clone and initiate

neoplastic transformation50,51

.

In megaloblastic anemia, due to folate deficiency there is reduction in

formation of methyl tetrahydrofolate. Its major biological action is to transfer

single carbon substituents to different compounds that is necessary for synthesis

of DNA. Lack of tetrahydrofolate leads to diminished synthesis of

deoxythymidylate monophosphate and consequently DNA. Due to impaired

DNA synthesis there is ineffective erythropoiesis characterized by the

appearance of morphologically abnormal nucleated red cell precursors called

megaloblasts. Megaloblasts are abnormal in function as well as in appearance,

18

with the result that the mature red cells formed from them are abnormal in size

and shape, the most prominent abnormality being macrocytosis. The term

megaloblastic macrocytic anemia describes the outstanding feature of both the

bone marrow and the peripheral blood52

.

In hypersplenism, the diagnostic criteria are enlargement of spleen,

peripheral blood cytopenia, normal or hypercellular bone marrow with normal

maturation and normalisation of blood cell count after splenectomy. The

pancytopenia results from sequestration of blood cells in enlarged spleen.

Normally about one third of total platelets in the body are pooled in the spleen,

enlarged spleen can sequester large number of platelets to induce

thrombocytopenia. A massively enlarged spleen can also trap a considerable

proportion of red cells and granulocytes to cause anemia and neutropenia

respectively53,54

.

In iron deficiency anemia, at the outset of chronic blood loss or other

states of negative iron balance, reserves in the form of ferritin and hemosiderin

may be adequate to maintain normal hemoglobin and hematocrit levels. There is

increased erythroid activity in the bone marrow. Anemia appears with

progressive depletion of iron stores. Red blood cells show microcytosis and

hypochromia. The white blood cells may be normal or mildly decreased.

Platelets are normal or sometimes increased as a reactive process. Iron

19

deficiency anemia usually presents with bicytopenia as anemia and

leukopenia55,56

.

Acute myeloid leukemia (AML), it is a tumor of hematopoietic

progenitors caused by acquired oncogenic mutations that impede differentiation.

It leads to the accumulation of immature myeloid forms (blasts) in the bone

marrow. The replacement of the marrow with blasts produce marrow failure and

complications related to anemia, thrombocytopenia and neutropenia. In

lymphocytic leukemias, the tumor cells suppresses normal hematopoiesis in the

bone marrow leading to pancytopenia. In multiple myeloma, there is

replacement of bone marrow cells by myeloma cells, suppression of

hematopoiesis and renal failure57,58

. In autoimmune disorders there is increased

peripheral destruction of blood cells in spleen and blood vessels.

In immune thrombocytopenia, auto antibodies of IgG type directed

against platelet membrane glycoproteins IIb-IIIa or Ib-IX complex. They act as

opsonins that are recognized by IgG Fc receptors expressed on phagocytes,

leading to increased platelet destruction in spleen. In some instances the

autoantibodies may bind to and damage megakaryocytes, leading to decrease in

platelet production. The principal changes are found in spleen, bone marrow and

peripheral blood59-62

.

Primary myelofibrosis, a hallmark feature is the development of

obliterative marrow fibrosis. The replacement of the marrow by fibrotic tissue

20

reduces bone marrow hematopoiesis leading to cytopenia and extensive

extramedullary hematopoiesis63

.

APPROACH TO PANCYTOPENIA

In pancytopenia both history and clinical findings play an important role

in diagnostic approach. The common clinical symptoms a patient present with

are easy fatigability, fever, bleeding manifestations, jaundice, abdominal pain,

bone pain, weight loss, etc. Pancytopenia presenting with any one or more of

the signs like splenomegaly, hepatomegaly, lymph node enlargement and bone

tenderness should help form a working diagnosis.

The hematological criteria for pancytopenia3 are

1. Hemoglobin less than 10 gm/dl

2. White blood cell counts less than 4000 cells/mm3

3. Platelet count less than 1,00,000/mm3

The important clue in complete blood count for differential diagnosis is

mean corpuscular volume (MCV). The normal value of MCV is 80-97 fl. It is

decreased in case of microcytic anemia and increased in case of macrocytic

anemia. In case of vitamin B12 and folate deficiency MCV value ranges from

97 -130 fl. Anemia with MCV of 97 to 110 fl may be related to other causes of

macrocytosis such as alcohol abuse, liver disease, hypothyroidism,

chemotherapeutic drugs and hematological disorders like hemolysis, aplastic

21

anemia and myelodysplastic syndromes64,65

. So in case of increased MCV, the

above causes must be ruled out before diagnosing megaloblastic anemia.

Coexisting iron deficiency produces dimorphic red cell patterns. Patients

with iron deficiency or thalassemia may have normal or low MCV, which fall

further after vitamin therapy. Iron deficiency sometimes blunts the erythroid

megaloblastic changes themselves morphologically. Iron studies in untreated

megaloblastic anemia often do not reveal the coexisting iron deficiency.

Because those marrow and blood indicators of iron status fall sharply within 24

to 48 hours of vitamin therapy, it is advisable to wait several more days for the

tests to stabilize in order to reveal the patient’s true iron status66,67

.

Peripheral blood film morphology is helpful to narrow down the

diagnosis. Bone marrow aspiration and biopsy were indispensable adjunct to

study the diseases of blood and the only way in which correct diagnosis can be

made. When performed correctly, they are simple and safest procedures that can

be repeated many times and can be performed on outpatients. The aspirated

material provides information about the numerical and cytological features of

marrow cells. Therefore, it has an important and complementary role in clinical

investigations and may have different relative merits in the assessment of

marrow disease.

In case of macrocytosis in peripheral blood, the presence of oval

macrocytic red cells and hypersegmented neutrophils are usually associated

22

with megaloblastic anemia. Bone marrow examination was done to differentiate

megaloblastic anemia from non-megaloblastic anemia particularly when MCV

values are not helpful. In megaloblastic anemia, the findings are panmyelosis

with morphologic hallmark is nuclear-cytoplasmic dissociation, which is best

appreciated in precursor cells. Megaloblastic nuclei are larger than normoblastic

nuclei and their chromatin appears abnormally dispersed due to its retarded

condensation (sieve-like or stippled chromatin). Giant band forms and

metamyelocytes with unusually large and often misshapen nuclei are typically

seen68-70

.

In case of iron deficiency anemia, the peripheral blood film shows

microcytic hypochromic cells. The zone of pallor is enlarged, hemoglobin may

be seen only in a narrow peripheral rim. Poikilocytosis in the form of small,

elongated red cells (pencil cells) is also seen. The bone marrow reveals cellular

marrow with increase in erythroid progenitors. A diagnostically significant

finding in the bone marrow is the absence of stainable iron in macrophages,

which is assessed by perl’s stain on aspirated marrow smears.

In peripheral blood film with very few or no blast and positive clinical

findings like hepatosplenomegaly and/or lymphadenopathy, myelodysplastic

disorder and subleukemic leukemia are considered as differential diagnosis. In

such cases examination of bone marrow indicate the diagnosis.

23

The hallmark of Myelodysplastic syndrome is presence of more than 10%

dysplasia in any one of the lineages. The findings in erythroid series include

erythroid hyperplasia with megaloblastoid features, nuclear budding,

multinucleation, karyorrhexis and cytoplasmic vacuole formation71

. The

myeloid series show dysplastic features like hyposegmentation of nucleus

(pseudo - Pelger Huet anomaly), reduced or absent granulation, ring-shaped

nuclei and abnormal localization of immature precursors in biopsy72,73

.

Abnormal megakaryocytes, including micromegakaryocytes, large mononuclear

forms, megakaryocytes with multiple dispersed nuclei and hypogranular

megakaryocytes are common bone marrow findings74

.

In leukemia, bone marrow aspiration smears reveal hypercellular marrow

with almost complete replacement of marrow by blast cells and reduced normal

hematopoiesis. Morphological features of various leukemias are identified in

bone marrow examination. In case of acute lymphoblastic leukemia,

lymphoblasts have more condensed chromatin, less conspicuous nucleoli and

smaller amount of cytoplasm that usually lacks granules. In acute myeloid

leukemia, myeloblasts have delicate nuclear chromatin, two to four nucleoli and

more voluminous cytoplasm. The cytoplasm contains fine peroxidase – positive

azurophilic granules and Auer rods, distinctive needle – like azurophilic

granules.

24

In immune thrombocytopenia, the peripheral blood reveals abnormally

large platelets (megathrombocytes). Blood loss may lead to anemia. On Bone

marrow examination, megakaryocytes are increased in size and are increased or

normal in number75,76

. Morphologic abnormalities of these giant cells are

present in most patients with IP. “Smooth” forms with single nuclei, scanty

cytoplasm, and relatively few granules are common77,78

. The findings are not

specific. The importance of doing bone marrow examination is to rule out

thrombocytopenia resulting from bone marrow failure or other primary bone

marrow disorders. Immune thrombocytopenia usually presents with bicytopenia

as anemia and thrombocytopenia.

In myelofibrosis, cellular phase and overtly fibrotic stages are present.

The bone marrow is hypercellular in cellular-phase and hypercellular or

hypocellular in fibrotic stage. In general, increased proliferation of granulocytic

and megakaryocytic lineage cells and reduced erythropoietic elements. The

bone marrow reveals clusters of atypical megakaryocytes with unusual nuclear

shapes (cloud-like nuclear morphology)79

.

In case of aplastic anemia, the red cells are normocytic normochromic,

few macrocytosis and mild anisocytosis with absence of red and white cell

precursors and usually reveals no positive findings on clinical examination like

hepatosplenomegaly and lymphadenopathy.

25

In case of multiple myeloma, the peripheral blood film show rouleaux

formation of red blood cells and bluish tinge in the background due to high level

of M proteins. If anemia present, it will be normocytic normochromic anemia,

total leucocyte count may be normal or low and platelet count is usually normal

or mildly reduced80

. The bone marrow examination reveals more than 10%

plasma cells. The plasma cells have a perinuclear clearing due to prominent

Golgi apparatus and eccentrically placed nucleus with clock face chromatin.

Sometimes plasmablasts and bizarre, multinucleated cells may be present.

Multiple myeloma usually presents as destructive plasma cell tumors involving

the axial skeleton as plasmacytomas.

26

WHO CLASSIFICATION OF THE MYELODYSPLASTIC

SYNDROMES81

(Revised in 2016)

NAME

DYSPLASTIC

LINEAGES

CYTO

PENIAS

RING

SIDERO

BLASTS

PB AND

BM

BLASTS

MDS with single

lineage dysplasia

(MDS-SLD)

1 1 or 2 <15% PB<1%,

BM<5%,

no Auer rods

MDS with

multilineage

dysplasia

(MDS-MLD)

2 or 3

1-3 <15% PB<1%,

BM<5%,

no Auer rods

MDS with ring

sideroblasts

(MDS-RS)

MDS-RS with

single

lineage dysplasia

(MDS-RS-SLD)

1 1 or 2 >15% PB<1%,

BM<5%,

no Auer rods

27

MDS-RS with

multilineage

dysplasia

(MDS-RS-MLD)

2 or 3 1-3 > 15% PB<1%,

BM<5%,

no Auer rods

MDS with isolated

del(5q)

1-3 1-2 None or

any

PB<1%,

BM<5%,

no Auer rods

MDS with excess

blasts(MDS-EB)

MDS-EB-1 0-3 1-3 None or

any

PB<2-4%,

BM<5-9%,

no Auer rods

MDS-EB-2 0-3 1-3 None or

any

PB<5-19%,

BM<10-

19%, no

Auer rods

28

MDS,

unclassifiable

(MDS-U)

With 1% blood

blasts

1-3 1-3 None or

any

PB=1%,

BM<5%,

no Auer rods

With single

lineage

dysplasia and

pancytopenia

1 3 None or

any

PB<1%,

BM<5%,

no Auer rods

based on defining

cytogenetic

abnormality

0 1-3 <15% PB<1%,

BM<5%,

no Auer rods

Refractory

cytopenia of

childhood

1-3 1-3 None PB<2%,

BM<5%,

29

Kishor Khodke et al82

studied 50 cases of pancytopenia for a period of 6

months. The inclusion criteria were Hemoglobin less than 10g/dl, total

leucocyte count less than 3.5×109/L and platelets less than 100×10

9/L. Bone

marrow aspiration done in all 50 cases along with trephine biopsy in 12 cases.

The age range was 3-69 years with male female ratio 1.3:1. Maximum number

of cases were in 12- 30 years of age. The common presenting illness was fever

(40%) and findings was pallor in all cases. Splenomegaly was seen in 40% of

cases and hepatomegaly was seen in 38% of cases. In peripheral smear,

hypersegmented neutrophils were seen in 40% of cases and dimorphic anemia

in 20%. The common cause was megaloblastic anemia (44%) followed by

aplastic anemia (14%) and kala-azar(14%).

B N Gayathri et al83

had done a prospective study for 2 years and studied

about 104 cases of pancytopenia. The study discussed in detail about age,

gender-wise incidence, presenting complaints, peripheral blood picture findings

and bone marrow examination findings. The inclusion criteria were hemoglobin

<9 g/dl, total leucocyte count <4000 cells/µl and platelet count <1,00,000/µl.

The only exclusion criteria was patients on myelotoxic chemotherapy. The age

of the patients ranged from 2 to 80 years. In adults, maximum number of cases

seen in the median age of 41 years with slight male predominance (M: F: 1.2:1).

In pediatric age groups, 31 cases had pancytopenia with slight female

predominance as opposed to adults. The most common cause of pancytopenia

30

was megaloblastic anemia (74.04%) followed by aplastic anemia (18.26%) and

subleukemic leukemia (3.85%). The most common clinical presentation was

generalized weakness and physical finding was pallor both of which seen in all

104 cases. Splenomegaly and hepatomegaly was observed in 35.57% and

26.92% of cases respectively. Dimorphic anemia (37.5%) followed by

macrocytic anemia (31.7%) was the predominant blood picture. In peripheral

smear, hypersegmented neutrophils noted in 51.35% cases of megaloblastic

anemia and relative lymphocytosis noted in 52.63% of cases in aplastic anemia.

The patients with megaloblastic anemia were treated with folic acid and vitamin

B12 who all showed complete clinical and hematological remission. The study

concluded that pancytopenia is a prognostic indicator in management of

patients.

Pathak et al5

studied 102 cases of pancytopenia in Nepal for a period of

one year. The inclusion criteria were Hemoglobin<10 g/dl, total leucocyte count

< 4×109/L and platelets less than 150×10

9/L. Bone marrow aspiration done in all

cases along with trephine biopsy was done in 48 cases. Maximum number of

cases was seen in age group of 15-30 years with slight female predominance.

The most common cause of pancytopenia was hypoplastic anemia (42.1%)

followed by hematological malignancies and megaloblastic anemia. In about 21

cases cause were remain undiagnosed, the bone marrow showed only erythroid

hyperplasia, eosinophilia and reactive myeloid hyperplasia. The study

31

concluded that bone marrow aspiration and biopsy can diagnose majority but

not all the cases of pancytopenia.

Arvind jain et al84

studied 250 cases of pancytopenia in Maharashtra for a

period of two years. The inclusion criteria were Hemoglobin < 13.3 g/dl for

men and <11.5 g/dl for women, Total leucocyte count <4 ×109/L and platelet

count <150 × 109/L. The maximum number of cases seen in 30 - 40 years with

male to female ratio of 2.6:1. The first cause of pancytopenia was

hypersplenism (29.2%), followed by infections (25.6%), myelosuppressants

(16.8%) and megaloblastosis (13.2%). The major cause of hypersplenism were

congestive splenomegaly and malaria. The cause of infections were AIDS and

septicemia. The study concluded that hypersplenism due to decompensated liver

disease and infections are on rise so it must be considered in cases of

Pancytopenia.

Sweta et al85

studied about 100 cases of pancytopenia. The inclusion

criteria were hemoglobin <10g/dl, total leucocyte count <4300 cells/µl and

platelet count <1,30,000/µl. The age range were 5 to 80 years with maximum

cases seen in 21 to 35 years of age and slight male predominance. The most

common cause was megaloblastic anemia (66%) followed by aplastic anemia

(18%) and malaria (6%). The study analyzed and recorded on range of

hemoglobin, total leucocyte count, platelet count, mean corpuscular volume

values and reticulocyte counts. The predominant blood picture was macrocytic

32

anemia (49%) followed by normocytic anemia (42%). The study found out that

82% cases of megaloblastic anemia was vegetarian. So that they must

supplement with vitamin B12 and folic acid drugs.

Akhtar Munir et al86

studied about 148 cases of pancytopenia/

bicytopenia in Pakistan to determine prevalence of non-malignant

hematological disorders. The study duration was three years. Maximum cases

are seen in age less than 10 years with slight female predominance. The

malignant hematological disorder was the most common cause (33.1%). Among

non-malignant hematological disorder, the most common was megaloblastic

anemia (18.2%) followed by idiopathic thrombocytopenic purpura (10.1%) and

iron deficiency anemia (9.5%) in non-malignant hematological disorders.

Mousa SM87

studied about 112 cases of pancytopenia/ bicytopenia in

adult Egyptian patients. The inclusion criteria were Hemoglobin< 13.3 g/dl for

men and <11.5 g/dl for women, Total leucocyte count <4×109/L and platelet

count <150 × 109/L. 50 cases of bicytopenia was studied and the most common

form was thrombocytopenia and anemia. The most common cause of

pancytopenia and bicytopenia was clonal hematopoietic disorders (34%). The

second common cause was hypersplenism (27%) in pancytopenia and idiopathic

thrombocytopenic purpura (24%) in bicytopenia. The third common cause was

aplastic anemia (21%) in pancytopenia and hypersplenism (18%) in

33

bicytopenia. The conclusion was the causes of cytopenia differs among

countries so bone marrow examination is an important investigation to be done.

Fahim manzoor et al88

studied 50 cases of pancytopenia in Srinagar for a

period of 2 years. The inclusion criteria were Hemoglobin<9 g/dl, total

leucocyte count less than 4×109/L and platelet count less than 140×10

9/L. The

maximum number of cases seen in between 21 - 30 years with slight male

predominance. The most common cause of pancytopenia was megaloblastic

anemia (56%), followed by hypoplastic/aplastic anemia (14%), hypersplenism

(8%) and post viral illness (6%). So that the megaloblastic anemia should

always be considered in evaluation of pancytopenia in Indian settings.

Shane Graham et al6 studied about the incidence of etiology of

pancytopenia. It is a prospective study done for one year. The number of cases

studied were 60 which included the patients of all age groups. The inclusion

criteria were Hemoglobin< 13.3 g/dl for men and <11.5 g/dl for women, Total

leucocyte count <4×109/L and platelet count <150 × 10

9/L. Patients received

previous blood transfusion and patients on chemotherapy/radiotherapy were

excluded from this study. They correlated clinical findings, hematological

indices and bone marrow study.The age of the patients ranged from 6 to 75

years with slight male predominance. Maximum number of cases were in the

age group of 30 to 55 years (70%). The most common cause was normoblastic

erythroid hyperplasia (30%) followed by megaloblastic anemia(20%), acute

34

myeloid leukemia (13.3%) and micronormoblastic maturation (10%). In this

study, it is suggested that cases with normoblastic erythroid hyperplasia

represent a phase in evolution of hypoplasia/aplasia. In this study they

calculated ratio of malignant etiology to non-malignant etiology as 1:4. The

conclusion of this study was that clinical and hematological examination is very

essential in planning management and prognosis of the pancytopenia patients.

Chetal Suva et al89

studied 50 cases of pancytopenia. The inclusion

criteria were Hemoglobin less than 10g/dl, total leucocyte count less than 4000

cells/µl and platelet count less than 1,50,000/µl. The age of the patients ranged

from 1 to 100 years with maximum number of cases seen in 11 to 20 years of

age and slight male predominance (3:2). The most common etiology was

megaloblastic anemia(50%) followed by aplastic anemia(38%) and

lymphoma(4%).

Bhagwan Singh Yadav et al90

studied 53 cases of pancytopenia in adults

for a period of one year. The inclusion criteria were Hemoglobin<10g/dl, total

leucocyte count < 4×109/L and platelet count less than 100×10

9/L.The mean age

of the patients are 35 ± 12 years with slight male predominance. The

commonest cause was megaloblastic anemia (35.84%) followed by septicemia

(11.32%) and alcoholic & non-alcoholic liver diseases(9.43% each).

Tariq Abdullah Mir et al91

studied about 132 cases of pancytopenia for a

period of 18 months. The inclusion criteria were Hemoglobin< 13.3 g/dl for

35

men and <11.5 g/dl for women, Total leucocyte count <4×109/L and platelet

count <150 × 109/L. The age of the patients ranged from 16 - 90 years with a

mean age of 43.81 years. The male to female ratio was 1.3: 1. The most

common cause of pancytopenia was megaloblastic anemia (72.73%) followed

by acute leukemia (6.81%) and multiple myeloma (5.3%). The conclusion was

in indian scenario, while evaluating the etiology of pancytopenia megaloblastic

anemia should always be kept in mind.

Kirti S. Dagdia et al3 studied about etiopathology of pancytopenia for a

period of 2 years and four months and total number of cases studied was 75. It

included patients of all ages and both sexes. The criteria were Hemoglobin less

than 10g/dl, total leucocyte count less than 4000 cells/µl and platelet count less

than 1,50,000/µl. They correlated clinical findings, hematological indices and

bone marrow examination. It was found that megaloblastic anemia (29.3%) was

the most common etiology of pancytopenia/bicytopenia in all age group

patients. Maximum number of patients were in the age group between 21 and 40

years with slight female predominance.The second cause was aplastic anemia

(18.6%). The most common presenting illness of patients with

pancytopenia/bicytopenia was generalized weakness (84%) and most common

clinical feature was pallor (100%). Splenomegaly was found in 20% of patients

and hepatomegaly in 13% of patients The conclusion was hematological indices

36

and bone marrow examination is a simple, economical and safe diagnostic tool

in evaluation of pancytopenia / bicytopenia patients.

Neelima bahal et al92

studied about various causes of pancytopenia and

bicytopenia. The number of cases studied were 129. The study recorded the

clinical history, examination findings, hematological, serological

and biochemical investigations. Hematological investigations done were

hematological indices, bone marrow aspiration and trephine biopsy. They have

done special stains like myeloperoxidase, periodic acid Schiff and reticulin stain

wherever required. The inclusion criteria was Hemoglobin<10g/dl, total

leucocyte count < 4×109/L and platelet count less than 100×10

9/L. The age

range of the patients were 7 months to 93 years with maximum number of cases

were in second to third decade. There is a slight male predominance in their

study. The study detailed about ranges of hemoglobin, total leucocyte count and

platelet count with percentage of patients involved in each group. The most

common form of bicytopenia was thrombocytopenia and anemia (57.97%)

followed by anemia and leucopenia (36.26%). The most common cause of

pancytopenia and bicytopenia were megaloblastic anemia involving 46.6% and

28.98% respectively. The second most common cause was leukemia involving

20% of pancytopenia patients and 23.18 % of bicytopenia patients. The study

interpreted that megaloblastic anemia and hypoplastic marrow commonly

present as pancytopenia and leukemia present as bicytopenia.

37

Pasam R et al93

studied 28 cases of pancytopenia. The inclusion criteria

were hemoglobin <10g/dl, total leucocyte count <3500 cell/µl and platelet

count<1,00,000/µl. The age range was 6 to 65 years with maximum cases seen

in second decade (28.7%). The commonest cause was viral fever (50%) which

improved after fever subsides. Two cases were improved with Vitamin B12 and

folic acid supplements. The conclusion of this study was to wait for at least 2-3

weeks in case of viral fever in which repeat hemogram improves after fever

subsides and also in cases of dimorphic blood picture supplementation with

vitamin B12 and folic acid improves blood counts. If pancytopenia persists, then

do bone marrow examination to rule out other causes like myelodysplastic

syndrome.

Ramdas jella et al94

studied about 56 cases of pancytopenia for a period of

2 years for the patients aged more than 18 years. The inclusion criteria were

Hemoglobin<10g/dl, total leucocyte count < 4×109/L and platelet count less

than 100×109/L. Males are affected more than females. The mean age of

patients presented with pancytopenia was 35 years. The first common cause of

pancytopenia was megaloblastic anemia (42.9%) and second was aplastic

anemia (23.2%).

Osman yokus et al95

studied 137 cases of pancytopenia in Istanbul for a

period of 2 years. The inclusion criteria were Hemoglobin<9 g/dl, total

leucocyte count < 4×109/L and platelet count less than 100×10

9/L. The average

38

age of patients affected are 63 years. In this study patients were divided into two

according to age,as patients less than 65 years (45cases) and more than 65 years

(92 cases). The most common cause of pancytopenia in patients aged more than

65 years was chronic liver disease (20%) followed by myelodysplastic

syndrome (19%). The most common cause in patients less than 65 years was

Vitamin B12deficiency (17%) and aplastic anemia (13%). The study concluded

that most of the causes of pancytopenia were associated with non-hematological

disorders and were diagnosed with laboratory investigations without the need

for bone marrow examination.

Anuja Dasgupta et al96

studied 80 cases of pancytopenia in Mangalore for

a period of 2 years. The inclusion criteria were Hemoglobin < 10 g/dl , Total

leucocyte count <4×109/L and platelet count <100 × 10

9/L. The age range was

2-90 years with maximum number of cases seen in 41-50 years and slight male

predominance. The most common cause was hypersplenism (28.75%), followed

by malaria (16.25%) and megaloblastic anemia (13.75%). The common cause

for hypersplenism was alcoholic liver disease in males and chronic liver disease

in females. Plasmodium vivax was common cause for malaria. The conclusion

was change in trends of the lifestyle and geographic location varies the causes

of pancytopenia.

Vikram singh et al97

studied about 214 cases of pancytopenia in medicine

and pediatrics department. A detailed physical examination, hematological and

39

biochemical investigations was done. The inclusion criteria were hemoglobin

< 8.5 g/dl, total leucocyte count < 3500 cells/ ul and platelet count < 1,00,000/

ul. The common cause of pancytopenia was aplastic anemia (36.9%) followed

by megaloblastic anemia (18.7%), kala-azar (11.7%) and myelodysplastic

syndrome (10.5%). An important observation in this study was difference in

serum LDH levels between megaloblastic anemia and myelodysplastic

syndrome. The serum LDH level in myelodysplastic syndrome was not more

than 410 IU/L while in megaloblastic anemia the lowest level was 1330

IU/L(ranges from 1330- 6550). The conclusion was megaloblastic anemia

should be considered as important cause of pancytopenia when serum lactate

dehydrogenase level is raised.

MATERIALS AND METHODS

40

MATERIALS AND METHODS

STUDY DESIGN

The present study is a Prospective study conducted in the Department of

Pathology during the period from November 2016 to June 2018. Ethical

clearance was obtained from the Ethics Committee of Coimbatore Medical

College, Coimbatore for the study.

The study sample included 150 cases of Pancytopenia/bicytopenia. For all

150 cases, complete blood count, peripheral smear and bone marrow

examination were analyzed for etiological diagnosis. Special stains like Sudan

Black B, Periodic Acid Schiff stain and Perl’s stain were done if needed.

PLACE OF STUDY:

Department of Pathology, Coimbatore Medical College, Coimbatore.

STUDY PERIOD:

November 2016 – June 2018

INCLUSION CRITERIA

1. Age - All ages

2. Gender - both male and female

3. Haemoglobin - less than 10 g/dl

4. Total leucocyte count less than 4000 cells/mm3

5. Platelet count less than 1,00,000 /mm3.

41

EXCLUSION CRITERIA

1. Patients on chemotherapy and radiotherapy.

2. Patients received blood transfusion.

Blood samples are received in ethylene diamine tetra-acetic acid (EDTA)

vacutainer. Detailed hematological investigations were done like complete

blood count, peripheral smear examination, reticulocyte count and erythrocyte

sedimentation rate. Measurement of hemoglobin, mean corpuscular volume,

total leucocyte count, differential count and platelet count was done on Sysmex

analyzer. They are cross-checked by peripheral smear examination. Peripheral

smears are made within 2 hours. Blood films are prepared in a clean glass slide

wiped free of dust using cotton. Slides should be 7.5 x 2.5 cm in size and

thickness of 1 mm. To facilitate labelling, one end is frosted. Spreader slide was

prepared by breaking the one corner of the slide by a glass cutter so its width is

1.8 cm. A spreader slide can be used repeatedly after thorough washing and

drying in between films2.

A drop of blood is placed at 1cm from one corner of a slide in the central

line and a spreader slide is placed at a 30degree angle in front of the drop. The

slide is then moved backwards so that it touches the drop. The drop spreads

quickly along the line of contact. Then the blood is spread along the slide and

the spreader slide should not be lifted till last drop of blood is spread. Ideal

smear should have a length of 3 cm and film should finishes 1cm before the end

42

of the slide. This forms the monolayer where the cells are widely spaced so that

cell counts can be made. Blood film made is allowed to air dry. The thickness of

the blood film can be regulated by varying the spreader angle or by changing

the spreading speed and pressure. For anemic blood, wider angle is used to

achieve correct thickness. For ideal thickness there should be some overlap of

red blood cells throughout the smear length. White blood cells and platelets

should be present throughout the blood film.

BUFFY COAT PREPARATION2

Buffy coat method was done for the concentration of abnormal cells or

white blood cells when they are in small numbers in peripheral blood in case of

cytopenias. Centrifuge an EDTA blood sample in a test tube for 5-10 minutes at

1200-1500 rpm. The red blood cells deposits down with buffy coat layer in

middle and plasma as supernatant. Remove the plasma with a pipette and then

deposit buffy coat layer on the slides by using the pipette. Mix with drop of

patient’s plasma and spread the smear. Allow it to air dry, fix and then stain

with usual stains.

STAINING BLOOD AND BONE MARROW FILMS

Romanowsky stains are employed universally for staining blood films.

Romanowsky dyes consists of two components 1. Azure B (Trimethyl thionin)

2. Eosin Y ( tetrabromo-fluorescein)98,99

. In Romanowsky group, Jenner is the

43

simplest and Giemsa is the complex dye. Routinely employed stain is the

Leishman stain. pH of the buffer recommended is 6.8. In order to obtain

uniform pH, 1 L of water is mixed with 50 ml of Sorensen’s phosphate buffer

for diluting the stain and for washing the slides100,101

.

LEISHMAN’S STAIN

Leishman stain was prepared by mixing 0.2 gm of Leishman powder with

100 ml of methanol in the conical flask. Warm the solution at 50degree celsius

for 15 minutes and then allow it to cool. Filtered solution can be used

immediately but the staining quality can be improved on standing for few hours.

After making blood films it is allowed to air dry. The slides are flooded

with Leishman stain for 2 minutes. Then added double the volume of buffered

water and kept for 5-7 minutes. Slides are washed in stream of buffered water

for 2 minutes so that it acquires a pinkish tinge. Back of the slide is washed and

set it upright for drying.

The mechanism by which different components of a cell's structure stain

with particular dyes depends on complex differences in binding of the dyes to

chemical structures and interactions between the dye molecules. Azure B is

bound to anionic molecules and eosin Y is bound to cationic sites on proteins102

.

Thus, the basic dye azure B uptaken by the acidic groupings of the

nucleic acids and proteins of the cell nuclei and primitive cytoplasm and

44

conversely, acidic dye eosin Y uptaken by the basic groupings on the

hemoglobin molecule. The granules in the cytoplasm of neutrophil leucocytes

are weakly stained by the azure complexes. Eosinophilic granules stains

strongly with the acidic component of the dye because it contains spermine

derivative, whereas basophilic granules stains strongly with the basic

component of the dye because it contains heparin. These effects depend on

molar equilibrium between the two dyes in time-dependent reactions. DNA

binds rapidly, RNA more slowly, and hemoglobin more slowly still. So it is

necessary to use the correct azure B to eosin ratio to avoid contamination and

for right time staining.

Bone marrow aspirates are usually done from iliac crest, sternum or

anterior or posterior iliac spines using Salah bone marrow aspiration needle103

.

The films are made for a length of 3-5 cm in a glass slide using a smooth- edged

glass spreader. After thorough drying, fix them and stain with Leishman stain. A

longer fixation time is essential for high quality staining. The films are stained

with Perl’s stain to demonstrate the presence or absence of iron wherever

necessary.

In cases of suspicion of leukemia, blood films made from buffy coat

preparation were stained with Leishman’s stain. Blood and bone marrow films

were stained with Sudan black B and Periodic Acid Schiff stains for

morphological typing of Acute Leukemias.

45

SUDAN BLACK B STAIN104

Sudan black B (SBB) is a lipophilic dye that binds irreversibly to

granule component in granulocytes, eosinophils and some monocytes.

REAGENTS USED :

1. Fixative- 40% formaldehyde solution.

2. Solution A - Mix Sudan Black B Powder 0.3gm and Ethanol 100ml,

shake frequently for 1-2 days and filter.

3. Solution B - Add pure phenol 16gms, ethanol 30 ml, Disodium phosphate

0.3 gm and distilled water 100ml and mix well.

4. Working solution - Add 12 ml of solution A after filtering to 8 ml of

solution B.

5. Leishman stain and buffer solution for counterstain.

PROCEDURE

1. Fixation with formalin.

2. Put the smear in working solution for 45 minutes.

3. Wash the smear in ethanol.

4. Rinse in tap water.

5. Air dry.

6. Pour Leishman stain for counterstaining and dilute with buffered water

for 20 minutes.

7. Wash in tap water.

8. Dry the smear.

46

RESULTS

The reaction product is black and granular. The most primitive

myeloblasts are negative. The granular positivity appears progressively as they

mature toward the promyelocyte stage. In granulocytic series, promyelocytes

and myelocytes are the most strongly staining cell with metamyelocytes and

neutrophils have progressively fewer positive granules.

PERIODIC ACID SCHIFF STAIN

Periodic acid specifically oxidizes 1–2 glycol groups to produce stable

dialdehydes. These dialdehydes give a red reaction product when exposed to

Schiff's reagent. In haemopoietic cells, the main source of positive reactions is

glycogen105

.

REAGENTS USED :

1. Periodic acid -0.5g in 100 ml of distilled water

2. Basic fuchsin -1gm

3. Sodium metabisulphite - 1.19 gm

4. 10N HCL - 0.75 ml

5. Activated charcoal - 0.5 gm

6. Distilled water - 100 ml

7. Haematoxylin

47

Preparation of Schiff’s reagent

Take 100ml of distilled water add 0.75 ml of 10N HCL then add 1 gm

of Basic fuchsin and 1.19gm of sodium metabisulphite and mix well. Keep it in

dark overnight in conical flask. Then add 0.5 gm of activated charcoal. After 15

minutes double filter the solution and keep it in refrigerator.

PROCEDURE

1. Fixation with formalin

2. Periodic acid - 5 minutes

3. Wash with tap water - 5 minutes

4. Schiff reagent - 45 minutes

5. Wash with water - 5 minutes

6. Haematoxylin - 30 minutes

7. Wash with water - 15 minutes

8. Differentiate with 1% acid alcohol

9. Wash in running water - 15 minutes

10. Dry the smear

48

RESULTS

The reaction product colour ranges from pink to bright red. Cytoplasmic

positivity may be diffuse or granular. Neutrophils show intense confluent

granular positivity. Normal erythroid precursors and red cells are negative.

Megakaryocytes and platelets show variable, diffuse positivity with

superimposed granules. Lymphocytes(10-40%) show granular positivity with

negative background cytoplasm106

.

PERL’S STAIN

The red cells contain granules of non-haem iron. The granules are

formed of a water-insoluble complex of ferric iron, lipid, protein, and

carbohydrate. The basis of a positive prussian blue reaction is the iron

containing material (or hemosiderin) reacts with potassium ferrocyanide to form

a blue compound, ferriferrocyanide107

.

REAGENTS USED :

1. 2% Hydrochloric acid

2. 2% Potassium ferrocyanide

3. 1% Eosin

49

PROCEDURE

1. Mix equal parts of 1 & 2

2. Keep the slide in the solution- 30 minutes

3. Wash the slide in running water 20 minutes to remove the sediment

4. Wash in distilled water

5. Counterstain with eosin for 1 minute

6. Wash in water

7. Dry the smear

RESULTS

The iron containing granules stain blue in a background of pink colour108

.

ANALYSIS OF PERIPHERAL SMEAR

Peripheral smear examination was done systematically under low, high

and oil immersion. In red blood cells, morphological changes like

anisopoikilocytosis, polychromasia, nucleated blood cells and Rouleaux

formation if present was analyzed. Anemias were classified according to

morphology into four types - normocytic normochromic, microcytic

hypochromic, macrocytic and dimorphic. Differential white blood cell count

was done and noted for any atypical and dysplastic changes. Platelet count and

50

morphology was analyzed. Peripheral smear examination was done to note the

presence of parasites if any.

SYSTEMATIC EXAMINATION OF BONE MARROW ASPIRATION

1. Stained films with marrow particles was assessed for the degree of

marrow cellularity as increased, normal or reduced in a low- power

objective(×10). As a rough guide, if haemopoietic cells constitute

less than 25% of the particle, it is hypocellular and if it was more

than 75-80%, it is hypercellular. The cellularity of the marrow is

affected by age. The proportion of fat cells to cellular marrow

particles are increased in adults than in children109,110

.

2. On low-power examination, megakaryocytes and clumps of non-

haemopoietic cells (e.g., metastatic carcinoma cells) were looked

for, mainly concentrated towards the tail of the film. In

megakaryocytes, number, morphology and maturation pattern were

examined in high power objective. Also examined morphology and

content of clumps if present. Looked for macrophages if any and its

morphology.

3. Myeloid erythroid ratio was calculated in a cellular area of the film

where the cells are well spread and stained.

51

4. The differential count for 200-500 cells were done and categorized

into erythroid, myeloid, lymphoid and plasma cells. The

morphology of these cells was also analyzed. In conditions, such as

leukemias and myelodysplastic syndrome, detailed differential

counts are important because the results may indicate prognosis

and affect treatment111

.

OBSERVATION AND

RESULTS

52

OBSERVATION AND RESULTS

TABLE 1: MEAN AGE OF THE STUDY

N Minimum Maximum Mean±SD

AGE 150 1 85 41.39 ± 17.6

TABLE 2: AGE DISTRIBUTION IN THE STUDY

Age in years Frequency Percent (%)

1-20 17 11.3

21-40 58 38.7

41-60 54 36.0

61-80 19 12.7

> 81 2 1.3

Total 150 100.0

53

CHART 1: AGE DISTRIBUTION IN THE STUDY

In this study, the minimum age of presentation was one year and

maximum age was 85 years. Most of the cases come under the age group of

21 – 40 years followed by 41 – 60 years. Mean age of the study was 41 years.

11%

39%36%

13%

1%

1-20 YEARS

21-40 YEARS

41-60 YEARS

61-80 YEARS

>81 YEARS

54

TABLE 3: GENDER DISTRIBUTION OF THE STUDY

Gender Frequency Percent (%)

Male 82 54.7

Female 68 45.3

Total 150 100.0

CHART 2: GENDER DISTRIBUTION IN THE STUDY

In this study, Males are involved more than females with a male to

female ratio of 1.2:1.

0

10

20

30

40

50

60

70

80

90

Male Female

82

68

Gender distribution

55

TABLE 4: MEAN WBC COUNT(×10³/mm³) OF THE STUDY

N Minimum Maximum Mean±SD

WBC(×10³/mm³) 150 0.6 11.0 3.60 ± 2.0

TABLE 5: MEAN HEMOGLOBIN (g/dl) OF THE STUDY

N Minimum Maximum Mean±SD

HB(g/dl) 150 1.9 10.0 6.02 ± 1.7

TABLE 6: MEAN PLATELET COUNT (×10³/mm³) OF THE STUDY

N Minimum Maximum Mean±SD

PLT(×10³/mm³) 150 3.0 282.0 6.02 ± 1.7

Table 7: Mean MEAN CORPUSCULAR VOLUME (fl) of the study

N Minimum Maximum Mean±SD

MCV(fl) 150 68.9 128.9 97.89 ± 13.97

56

CHART 3: MEAN LABORATORY VALUES

In this study, the mean value of White blood cell count- 3.6×10³/mm³,

Hemoglobin- 6g/dl , Platelet count-60,540/mm³ and Mean Corpuscular volume-

97.89fl are in the reference range defined for haematological criteria.

0

10

20

30

40

50

60

70

80

90

100

WBC(×10³/mm³) HB(g/dl) PLT(×10³/mm³) MCV(fl)

3.6076.025

60.54

97.895

Mean laboratory values

57

TABLE 8:MEAN WBC COUNT IN PANCYTOPENIA VERSUS

BICYTOPENIA

Peripheral smear N Mean

Std.

Deviation

P

Value

WBC

(×10³/mm³)

PANCYTOPENIA 101 2.585 .8023

.000* BICYTOPENIA 49

5.714 2.1282

CHART 4: MEAN WBC COUNT IN PANCYTOPENIA VS

BICYTOPENIA

0

1

2

3

4

5

6

Pancytopenia Bicytopenia

2.585

5.714

58

TABLE 9:MEAN HB VALUE IN PANCYTOPENIA VERSUS

BICYTOPENIA

Peripheral smear N Mean

Std.

Deviation

P

Value

HB(g/dl)

PANCYTOPENIA 101 5.773 1.7860

.011*

BICYTOPENIA 49 6.545 1.6783

CHART 5: MEAN HB VALUE IN PANCYTOPENIA VS BICYTOPENIA

5.2

5.4

5.6

5.8

6

6.2

6.4

6.6

Pancytopenia Bicytopenia

5.773

6.545

59

TABLE 10:MEAN PLATELET COUNT IN PANCYTOPENIA VERSUS

BICYTOPENIA

Peripheral smear N Mean

Std.

Deviation

P

Value

PLT(×10³/mm³)

PANCYTOPENIA 101 50.743 24.6814

.000*

BICYTOPENIA 49 80.735 50.1547

CHART 6: MEAN PLATELET COUNT IN PANCYTOPENIA VERSUS

BICYTOPENIA

0

10

20

30

40

50

60

70

80

90

Pancytopenia Bicytopenia

50.743

80.735

60

TABLE 11:MEAN MCV VALUE IN PANCYTOPENIA VERSUS

BICYTOPENIA

Peripheral smear N Mean

Std.

Deviation

P

Value

MCV(fl)

PANCYTOPENIA 101 99.873 14.0581

.012* BICYTOPENIA 49 93.816 13.0254

*-STATISTICALLY SIGNIFICANT (P<0.05)

CHART 7: MEAN MCV VALUE IN PANCYTOPENIA VS

BICYTOPENIA

In this study, the mean value of WBC count, Hemoglobin and

Platelet count in pancytopenia is less than that of bicytopenia which was

statistically significant. It implies that severity of anemia, leucopenia and

thrombocytopenia is high in case of pancytopenia. The mean MCV value is high

in pancytopenia and normal in bicytopenia which implies macrocytic anemia is

more common in pancytopenia than bicytopenia.

90

91

92

93

94

95

96

97

98

99

100

Pancytopenia Bicytopenia

99.873

93.816

61

TABLE 12: PERIPHERAL SMEAR DIAGNOSIS

Peripheral Smear

Diagnosis Frequency Percent (%)

BICYTOPENIA 49 32.7

PANCYTOPENIA 101 67.3

Total 150 100.0

CHART 8: PERIPHERAL SMEAR DIAGNOSIS

33%

67%

BICYTOPENIA

PANCYTOPENIA

62

TABLE 13: TYPE OF ANEMIA IN PERIPHERAL SMEAR

Type of Anemia Frequency Percent (%)

ACUTE LEUKEMIA 2 1.3

DIMORPHIC ANEMIA 75 50.0

MACROCYTIC ANEMIA 57 38.0

MICROCYTIC HYPOCHROMIC

ANEMIA 10 6.7

NORMOCYTIC NORMOCHROMIC

ANEMIA

4 2.7

SUBLEUKEMIC LEUKEMIA 2 1.3

Total 150 100.0

CHART 9: TYPE OF ANEMIA IN PERIPHERAL SMEAR

0

20

40

60

80

Acu

te L

eu

ke

mia

Dim

orp

hic

An

em

ia

Ma

cro

cyti

c A

ne

mia

Mic

rocy

tic

Hy

po

chro

mic

An

em

ia

No

rmo

cyti

c N

orm

och

rom

ic A

ne

mia

Su

ble

uk

em

ic L

eu

ke

mia

2

75

57

104 2

63

In this study, out of 150 cases of pancytopenia/bicytopenia dimorphic

anemia is the most common presentation followed by macrocytic anemia. The

dimorphic anemia may be a combination of macrocytes and microcytic

hypochromic cells or microcytic hypochromic cells and normocytic

normochromic cells or macrocytes and normocytic normochromic cells. In this

study, combination of microcytes and macrocytes is the most common form of

dimorphic anemia.

In leukemia cases, 2 cases presented with more than 20% blasts in

peripheral smear termed as acute leukemia and 2 cases presented with less than

5% blasts termed as subleukemia leukemia. For these 2 cases, buffy coat slide

was prepared and then stained with leishman's stain which showed 10% blasts.

64

TABLE 14: TYPE OF ANEMIA IN PANCYTOPENIA VS

BICYTOPENIA

TYPE OF ANEMIA

PERIPHERAL SMEAR

PANCYTOPENIA BICYTOPENIA

ACUTE LEUKEMIA 1 (1.0%) 1 (2.0%)

DIMORPHIC ANEMIA 43 (42.6%) 32 (65.3%)

MACROCYTIC ANEMIA 46 (45.5%) 11 (22.4%)

MICROCYTIC

HYPOCHROMIC ANEMIA

6 (5.9%) 4 (8.2%)

NORMOCYTIC

NORMOCHROMIC

ANEMIA

3 (3.0%) 1 (2.0%)

SUBLEUKEMIC

LEUKEMIA

2 (2.0%) 0 (0.0%)

65

CHART 10: TYPE OF ANEMIA IN PANCYTOPENIA VS

BICYTOPENIA

In this study, when cases are divided into pancytopenia and

bicytopenia, macrocytic anemia became the most common one in pancytopenia

and dimorphic anemia in bicytopenia.

0

5

10

15

20

25

30

35

40

45

50

Acu

te L

eu

ke

mia

Dim

orp

hic

An

em

ia

Ma

cro

cyti

c A

ne

mia

Mic

rocy

tic

Hy

po

chro

mic

An

em

ia

No

rmo

cyti

c N

orm

och

rom

ic A

ne

mia

Su

ble

uk

em

ic L

eu

ke

mia

1

43

46

6

3 21

32

11

4

1 0

Pancytopenia

Bicytopenia

66

TABLE 15: BONE MARROW DIAGNOSIS

Bone Marrow Findings Frequency Percent (%)

ACUTE LEUKEMIA 6 4.0

IMMUNE

THROMBOCYTOPENIA

4 2.7

COMBINED

DEFICIENCY

63 42.0

MEGALOBLASTIC

ANEMIA

57 38.0

METASTASIS 3 2.0

MICRONORMOBLASTIC

ERYTHROID

HYPERPLASIA

8 5.3

MYELO FIBROSIS 2 1.3

MYELODYSPLASTIC

SYNDROME

6 4.0

PLASMACYTOMA 1 .7

Total 150 100.0

67

CHART 11: BONE MARROW DIAGNOSIS

In this study, the most common diagnosis is combined deficiency

(megaloblastic and micronormoblastic maturation) in 150 cases of

pancytopenia/ bicytopenia followed by megaloblastic anemia.

0

10

20

30

40

50

60

70A

cute

Le

uk

em

ia

Imm

un

e T

hro

mb

ocy

top

en

ia

Co

mb

ine

d D

efi

cie

ncy

Me

ga

lob

last

ic A

ne

mia

Me

tast

asi

s

Mic

ron

orm

ob

last

ic E

ryth

roid

Hy

pe

rpla

sia

My

elo

Fib

rosi

s

My

elo

dy

spla

stic

Sy

nd

rom

e

Pla

sma

cyto

ma

64

63

57

3

8

26

1

68

TABLE 16: BONE MARROW DIAGNOSIS IN PANCYTOPENIA

VERSUS BICYTOPENIA

BONE MARROW DIAGNOSIS PANCYTOPENIA BICYTOPENIA

ACUTE LEUKEMIA 3 (3.0%) 3 (6.1%)

IMMUNE

THROMBOCYTOPENIA

0 (0.0%) 4 (8.2%)

COMBINED DEFICIENCY 42 (41.6%) 21 (42.9%)

MEGALOBLASTIC ANEMIA 46 (45.5%) 11 (22.4%)

METASTASIS 1 (1.0%) 2 (4.1%)

MICRONORMOBLASTIC

ERYTHROID HYPERPLASIA

5 (5.0%) 3 (6.1%)

MYELOFIBROSIS 2 (2.0%) 0 (0%)

MYELODYSPLASTIC

SYNDROME

2 (2.0%) 4 (8.2%)

PLASMACYTOMA 0 (0.0%) 1 (2.0%)

69

CHART 12 : BONE MARROW DIAGNOSIS IN PANCYTOPENIA

VERSUS BICYTOPENIA

In this study, when cases are divided into pancytopenia and

bicytopenia, megaloblastic anemia became the most common diagnosis in

pancytopenia and combined deficiency in bicytopenia cases.

0

5

10

15

20

25

30

35

40

45

50

Acu

te L

eu

ke

mia

Imm

un

e T

hro

mb

ocy

top

en

ia

Co

mb

ine

d D

efi

cie

ncy

Me

ga

lob

last

ic A

ne

mia

Me

tast

asi

s

Mic

ron

orm

ob

last

ic E

ryth

roid

Hy

pe

rpla

sia

My

elo

Fib

rosi

s

My

elo

dy

spla

stic

Sy

nd

rom

e

Pla

sma

cyto

ma

30

42

46

1

52 2

03 4

21

11

2 30

41

Bone marrow diagnosis in pancytopenia and bicytopenia

Pancytopenia

Bicytopenia

COLOUR PLATES

COLOUR PLATES

MEGALOBLASTIC ANEMIA

Figure 1 : MACROCYTES in peripheral smear

( Leishman stain - Oil Immersion)

Figure 2 : HYPERSEGMENTED NEUTROPHIL

in peripheral smear ( leishman stain - oil immersion)

Figure 3 : MEGALOBLASTS AND GIANT BAND FORMS

in bone marrow aspirate ( Leishman stain - Oil Immersion)

DIMORPHIC ANEMIA

Figure 4 : MACROCYTES AND MICROCYTES

in peripheral smear ( Leishman stain - Oil Immersion)

Figure 5 : MICRONORMOBLASTS AND MEGALOBLASTS

in bone marrow aspirate ( Leishman stain - Oil Immersion)

Figure 6 : MICROCYTIC HYPOCHROMIC ANEMIA

in peripheral smear ( Leishman stain - Oil Immersion)

Figure 7 : MICRONORMOBLASTS

in bone marrow aspirate ( Leishman stain - Oil Immersion)

IMMUNE THROMBOCYTOPENIA

Figure 8 : GIANT PLATELET

in peripheral smear (Leishman stain - Oil Immersion)

Figure 9 : HYPOLOBATED MEGAKARYOCYTE

in bone marrow ( Leishman stain - Oil Immersion)

MYELODYSPLASTIC SYNDROME

Figure 10 : DYSPLASTIC ERYTHROID LINEAGE

in bone marrow ( Leishman stain - Oil Immersion)

Figure 11 : DYSPLASTIC NEUTROPHILS

in bone marrow ( Leishman stain - Oil Immersion)

DYSPLASTIC MEGAKARYOCYTES

Figure 12.1 Megakaryocyte with multiple dispersed nuclei

Figure 12.2 : Hypolobated and mononuclear forms

in bone marrow aspirate ( Leishman stain - Oil Immersion)

Figure 13 : ACUTE LYMPHOBLASTIC LEUKEMIA

in bone marrow aspirate ( Leishman stain - Oil Immersion)

Figure 14 : ACUTE MYELOID LEUKEMIA

in peripheral smear ( Leishman stain - Oil Immersion)

Figure 15 : ACUTE MYELOID LEUKEMIA

in bone marrow aspirate ( Leishman stain - Oil Immersion)

Figure 16 : SUDAN BLACK B POSITIVE MYELOBLASTS

(Oil Immersion)

Figure 17 : SUBLEUKEMIC LEUKEMIA –BUFFY COAT SMEAR

( Leishman stain - Oil Immersion)

Figure 18 : METASTATIC DEPOSITS

in bone marrow aspirate ( Leishman stain - Oil Immersion)

PLASMACYTOMA

Figure 19 : ROULEAUX FORMATION

in peripheral smear ( Leishman stain - Oil Immersion)

Figure 20 : PLASMA CELLS

in bone marrow aspirate ( Leishman stain - Oil Immersion)

DISCUSSION

70

DISCUSSION

The incidence of pancytopenia / bicytopenia is increasing in frequency

due to multifactorial causation. Pancytopenia/ bicytopenia are becoming the

common hematological findings with variable clinical manifestations. It became

a challenging one for the clinicians to diagnose the correct etiology for the

management of the patients. The causes of pancytopenia/ bicytopenia are many

diseases which are diagnosed by doing complete hematological profile,

peripheral smear and bone marrow study. The current study was done to

evaluate the etiological diagnosis by analyzing hematological indices, peripheral

smear and bone marrow aspiration study for 150 cases of pancytopenia/

bicytopenia.

AGE AND SEX

The age of presentation ranged from 1 year to 85 years. The maximum

number of cases was seen in the age group of 21 - 40 years. Males (54.7%) are

more commonly involved than females (45.3%) with a male to female ratio of

1.2:1. The results are similar to the study done by Gayathri et al83

and Neelima

bahal et al92

but in contrast to Kirti S Dagdia et al³ which showed slight female

predominance.

HEMOGRAMS

In the present study, the hematological criteria for pancytopenia were

hemoglobin less than 10 gm/dl, total white blood cell count less than 4000

71

cells/mm³ and platelet count less than 1,00,000/mm³. In cases of bicytopenia

any two of the above criteria with other value being normal. So the range of

each parameter ranged from low to normal values. In 150 cases, there were 101

cases of pancytopenia and 49 cases of bicytopenia. The hemoglobin value

ranged from 1.9 to 10 gm/dl, the total count ranged from 600 to 11,000

cells/mm³ and the platelet count ranged from 3000 to 2,82,000/mm³.

The mean value of hemoglobin, white blood cell count and platelet

count are low in cases of pancytopenia than bicytopenia. So the severity of

anemia, leucopenia and thrombocytopenia are high in case of pancytopenia than

bicytopenia which are statistically significant. The mean corpuscular volume

ranged from 68.9 to 128.9 fl. The mean value of MCV is 93.8 in cases of

bicytopenia and 99.8 in cases of pancytopenia which shows MCV is increased

in pancytopenia. The increase in MCV indicates macrocytic anemia is high in

pancytopenia cases. The normal MCV indicates normocytic normochromic

anemia and dimorphic anemia are high in cases of bicytopenia.

PERIPHERAL SMEAR DIAGNOSIS

In total of 150 cases, the most common anemia in peripheral smear is

dimorphic anemia in 75 cases (50%), followed by macrocytic anemia in 57

cases (38%). But in pancytopenia cases (101 cases) the most common anemia is

macrocytic anemia in 46 cases (45.5%) followed by dimorphic anemia in 43

cases (42.6%). In bicytopenia (49 cases), the most common anemia is dimorphic

72

anemia in 32 cases (65.3%) followed by macrocytic anemia in 11 cases

(22.4%). Acute leukemia and subleukemic leukemia constitute 2 cases each

(2.6%). Bone marrow aspiration study yielded the final etiological diagnosis as

follows.

CAUSES OF PANCYTOPENIA

The most common etiology of pancytopenia is megaloblastic anemia in

46 cases (45.5%). The second common is combined deficiency with

megaloblastic and micronormoblastic maturation in 42 cases (41.5%)

followed by micronormoblastic erythroid hyperplasia (5%), leukemia (3%),

myelodysplastic syndrome (2%), myelofibrosis (2%) and metastatic

deposits (1%).

CAUSES OF BICYTOPENIA

The most common etiology of bicytopenia is combined deficiency with

megaloblastic and micronormoblastic maturation in 21 cases (42.8%). The

second common is megaloblastic anemia in 11 cases (22.4%) followed by

myelodysplastic syndrome (8.2%), immune thrombocytopenia (8.2%), leukemia

(6.1%), micronormoblastic erythroid hyperplasia (6.1%), metastasis (4.1%) and

plasmacytoma (2%).

73

TABLE 17 : COMPARISON OF VARIOUS ETIOLOGY OF

PANCYTOPENIA/BICYTOPENIA IN DIFFERENT STUDIES

STUDIES

PANCYTOPENIA BICYTOPENIA

FIRST

CAUSE

SECOND

CAUSE

FIRST

CAUSE

SECOND

CAUSE

Mousa

SM87

;

Egypt 2014

Clonal

hematopoietic

disorders

(34%)

Hypersplenism

(27.4%)

Clonal

hematopoietic

disorders (34%)

Immune

thrombocytopenia

(24%)

Akhtar

Munir et

al86

;

Pakistan

2014

Malignant

hematological

disorders

(33.1%)

Megaloblastic

anemia

(18.2%)

Malignant

hematological

disorders

(33.1%)

Megaloblastic

anemia (18.2%)

Neelima

Bahal et

al92

; India

2016

Megaloblastic

anemia

(46.6%)

Leukemia

(20%)

Megaloblastic

anemia

(28.98%)

Leukemia

(23.18%)

Kirti S

Dagdia et

al3; India

2016

Megaloblastic

anemia

(29.3%)

Aplastic

anemia

(18.6%)

Megaloblastic

anemia (29.3%)

Aplastic anemia

(18.6%)

Present

study

Megaloblastic

anemia

(45.5%)

Combined

deficiency

(41.5%)

Combined

deficiency

(42.8%)

Megaloblastic

anemia (22.4%)

74

MEGALOBLASTIC ANEMIA

In the present study, 45.5% cases of pancytopenia and 22.4% cases of

bicytopenia were diagnosed as megaloblastic anemia. Various studies done by

Kirti S Dagdia et al³, Kishore khodke et al82

, Gayathri et al83

and Sweta et al85

also showed megaloblastic anemia as most common cause of pancytopenia. But

in contrast, the other study done by Mousa SM87

showed clonal hematopoietic

disorders as most common etiology in cases of pancytopenia and bicytopenia.

The MCV is increased in both cases but more in case of pancytopenia upto

128.9 fl due to severity of anemia. The peripheral smear showed macrocytes

and macroovalocytes in all cases with hypersegmented neutrophils in most of

the cases. The bone marrow aspiration showed cellular marrow with erythroid

hyperplasia and predominance of erythroid precursors showing sieve like

chromatin. Giant band forms, metamyelocytes and hypersegmented neutrophils

are seen in all cases of megaloblastic anemia. Few of the cases show dysplastic

features in erythroid precursors like nuclear budding and binucleation which

constitutes less than 5%. It is important because if it is more than 10%, then

myelodysplastic syndrome must be ruled out.

COMBINED DEFICIENCY

In the present study, 42.8% cases of bicytopenia and 41.5% cases of

pancytopenia were diagnosed as combined deficiency with megaloblastic and

micronormoblastic maturation. In a study done by Kibria et al¹¹² showed

75

combined deficiency anemia as the most common etiology of pancytopenia

(24.87%). Kirti S Dagdia et al³ showed combined deficiency as third most

common etiology of pancytopenia/bicytopenia in 12% cases The MCV value is

normal in both cases but high normal in cases of pancytopenia. The peripheral

smear showed dimorphic anemia composed of macrocytes, macroovalocytes

and microcytic hypochromic cells. The predominant of which cell type varies

with type of deficiency, in case of increased deficiency of iron than vitamin B12

and folic acid, the microcytic hypochromic cells are predominant than

macrocytes and vice versa in case of increased vitamin B12 and folic acid

deficiency. It is because of high prevalence of nutritional deficiency in India,

combined deficiency is increasing.

In our hospital, most of the patients were from low socioeconomic

status, so they presents with deficiency of both iron and vitamin B12 and folic

acid. The bone marrow of combined deficiency anemia showed a cellular

marrow with erythroid hyperplasia having megaloblasts and micronormoblasts.

The combined deficiency presented with bicytopenia more than pancytopenia in

the present study. It is because in case of iron deficiency, platelets are increased

as reactive process so that patients with combined deficiency compensates the

increase in platelets and presents as bicytopenia with anemia and leucopenia as

common findings with normal platelet count.

76

MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

In the present study, 6.1% cases of bicytopenia and 5% cases of

pancytopenia were diagnosed as micronormoblastic erythroid hyperplasia. The

study done by Akhtar Munir et al86

had 9.5% cases of iron deficiency anemia. In

case of iron deficiency, microcytic hypochromic anemia is a common finding.

Leucopenia may be present. But as a reactive process, increased erythropoietin

stimulates megakaryopoiesis so that platelet count may be normal or increased.

In cases of severe iron deficiency, the patient presents with pancytopenia. In the

present study, many cases of microcytic hypochromic anemia presented with

bicytopenia than pancytopenia accordingly.

MYELODYSPLASTIC SYNDROME

In this study, 8.2% cases of bicytopenia and 2% cases of pancytopenia

were diagnosed as myelodysplastic syndrome (MDS). Kirti S Dagdia et al³ had

8% cases of MDS, Kishore Khodke et al82

had 2% cases of MDS and Vikram

Singh et al97

had 10.7% cases of MDS. Myelodysplastic syndrome classified

into many subtypes according to cytopenias and dysplastic lineages. As per

2016 revised WHO classification, out of 4 cases of bicytopenia 3 cases

diagnosed as MDS with multilineage dysplasia and 1 case as MDS with single

lineage dysplasia. 2 cases of pancytopenia diagnosed as MDS with multilineage

dysplasia. The MDS presented with dimorphic anemia in 4 cases, microcytic

hypochromic anemia in 1 case and normocytic normochromic anemia in 1 case.

77

Bone marrow aspiration study showed more than 10% dysplastic features in

erythroid, myeloid and megakaryocytic lineages. Ringed sideroblasts are not

seen in perl’s stain.

IMMUNE THROMBOCYTOPENIA

In the present study, 8.2% cases of bicytopenia were diagnosed as

immune thrombocytopenia. Akhtar Munir et al86

had 10.1% cases and Mousa

SM87

had 24% cases of immune thrombocytopenia as second most common

etiology of bicytopenia. Immune thrombocytopenia usually presents as

thrombocytopenia, but our cases presented with anemia and thrombocytopenia.

It is because of chronic bleeding in the patients leads to anemia. The peripheral

smear findings are dimorphic anemia with normocytic normochromic cells and

microcytic hypochromic cells and thrombocytopenia with giant platelets. The

bone marrow aspiration showed increased megakaryocytes with many

hypolobated and hypogranular forms.

ACUTE LEUKEMIA

In this study, 3 cases (3%) of pancytopenia and 3 cases (6.1%) of

bicytopenia were diagnosed as acute leukemia. Kirti S Dagdia et al³ had 17.3%

cases of leukemia, Kishore Khodke et al82

and Sweta et al85

had 2% cases of

acute leukemia. In pancytopenia cases, two of them presented as subleukemic

leukemia in peripheral smear and confirmed the diagnosis by bone marrow

aspiration study. In bicytopenia cases, two of them were diagnosed only after

78

bone marrow aspiration study which showed 30 – 40 % blasts, as peripheral

smear showed no blasts. In subleukemic leukemia cases, buffy coat was

prepared and then stained with cytochemical stains for morphological diagnosis.

Bone marrow aspiration study showed hypercellular marrow with 70 - 80%

blasts. Out of 6 cases, 4 cases were diagnosed as acute lymphoblastic leukemia

morphologically subtyped as ALL-L2, all of which are seen in children and

adolescents and 2 were diagnosed as acute myeloid leukemia morphologically

typed as acute promyelocytic leukemia (APML), both of which are seen in

young adults.

METASTASIS

In the present study, 1 case (1%) of pancytopenia and two cases (4.1%)

of bicytopenia were diagnosed as metastatic deposits. Kirti S Dagdia et al³ had

1.3% cases and Neelima bahal et al92

had 1.5 % cases of metastasis to bone

marrow. The peripheral smear showed normocytic normochromic anemia. Bone

marrow aspiration showed atypical epithelial cells.

MYELOFIBROSIS

In this study, 2 cases (2%) of pancytopenia were diagnosed as

myelofibrosis. Neelima Bahal et al82

and Vikram Singh et al97

had 2 cases of

myelofibrosis. The peripheral smear showed normocytic normochromic anemia.

The bone marrow aspiration showed hypocellular marrow with increased

megakaryocytes which are in clusters and abnormal cloud like forms.

79

PLASMACYTOMA

In this study, only 1 case (2%) of bicytopenia diagnosed as

plasmacytoma. Kirti S Dagdia et al³ also had 1 case (1.3%) of plasmacytoma.

Kishore Khodke et al82

, Sweta et al85

and Vikram Singh et al97

had 2 cases of

plasmacytoma. The peripheral smear showed dimorphic anemia with rouleaux

formation. The bone marrow aspiration showed more than 30% plasma cells

and its immature forms.

CONCLUSION

80

CONCLUSION

As pancytopenia and bicytopenia is increasing in frequency, it is

important to evaluate the various etiologies for the management of patients.

Many studies were done on pancytopenia but for bicytopenia very limited

number of studies available. In this study, causes for bicytopenia also evaluated

as it is equally important as pancytopenia in the management of patients. In this

study, 150 cases were studied with maximum number of patients were in the

age group of 21-40 years with slight male predominance.

The most common cause of pancytopenia was megaloblastic anemia

followed by combined deficiency whereas for bicytopenia it was combined

deficiency followed by megaloblastic anemia. The causes of cytopenias differ

between countries according to health problems which is prevalent there. In

other countries hematological malignancies are the most common cause of

pancytopenia / bicytopenia. The higher incidence of combined deficiency in our

country can be attributed to low socioeconomic status, inadequate nutrition,

poor hygiene and lifestyle modification. So analysis of hematological indices,

peripheral smear and bone marrow study are very important for an early

intervention for to enhance the survival rate for the patients.

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81

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ANNEXURES

I: MASTER CHART

S.NO H NO AGE SEX WBC(×10³/mm³) HB(g/dl) PLT(×10³/mm³) MCV(fl)PERIPHERAL SMEAR

DIAGNOSISTYPE OF ANEMIA BONE MARROW FINDINGS

1 H1122/16 55 F 3.1 3.2 24 123.2 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

2 H1161/16 48 M 1.8 1.9 25 120 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

3 H1176/16 73 F 1.8 7.2 29 113.2 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

4 H1195/16 16 F 2.2 6.4 20 88.3 PANCYTOPENIA SUBLEUKEMIC LEUKEMIA ACUTE LEUKEMIA

5 H1196/16 49 M 1.2 2.9 44 112.2 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

6 H1197/16 45 F 3.7 7.2 55 95.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

7 H1259/16 37 M 2.4 5.4 27 86.3 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

8 H1277/16 47 M 2.4 4.9 28 106.9 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

9 H1281/16 73 F 8.6 6.9 26 91.3 BICYTOPENIA DIMORPHIC ANEMIA MYELODYSPLASTIC SYNDROME

10 H1294/16 44 M 3.6 7.6 282 106 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

11 H1304/16 50 F 1.6 10 82 79 PANCYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

12 H1317/16 60 F 5.8 6.6 99 109 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

13 H1342/16 6 F 6.2 8.5 95 82 BICYTOPENIA NORMOCYTIC NORMOCHROMIC ANEMIA METASTASIS

14 H1359/16 2 F 4.5 4 69 84.8 BICYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA ACUTE LEUKEMIA

15 H1381/16 45 M 3.2 5.6 38 107.2 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

16 H1394/16 60 F 3 9 40 71 PANCYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

17 H1424/16 16 F 2.4 4.6 59 118.4 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

18 H1433/16 30 M 4.7 7.2 62 98.6 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

19 H1435/16 33 M 3.4 4.9 68 109.3 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

20 H1467/16 38 M 4.2 3.1 74 92 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

21 H1534/16 63 M 2 4 31 102 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

22 H1541/16 31 F 4.9 4 99 95.9 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

23 H1549/16 25 F 2 6.5 91 103.2 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

24 H1567/16 25 F 2.7 4 89 100.3 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

25 H1640/16 63 M 3.3 8.1 38 105.4 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

26 H1642/16 40 M 2.5 2.5 38 108.1 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

27 H1678/16 40 M 1.7 3.1 55 88.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

28 H1693/16 28 M 3.2 4.5 80 92.9 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

29 H1708/16 27 M 4.8 5.5 50 112.8 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

30 H1724/16 37 M 3.4 3.8 8 108.6 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

31 H1749/16 55 F 3.3 5.8 64 116.2 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

32 H1783/16 46 F 2.4 5.9 44 121.2 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

33 H98/17 52 F 2.8 4.9 90 104.5 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

34 H122/17 50 M 3.5 4.9 45 98.4 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

35 H123/17 28 F 1.3 3.4 27 91.7 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

36 H175/17 23 M 3.9 3.4 36 101 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

37 H195/17 60 F 1.2 5.5 34 106 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

38 H204/17 57 M 2.7 5 83 97.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

39 H291/17 55 M 2.5 5.3 28 116.7 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

40 H292/17 25 F 1.7 4.6 24 88.2 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

41 H324/17 72 M 3.5 3.6 44 124.1 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

42 H329/17 16 M 4.9 6.4 89 104.3 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

43 H471/17 80 F 2.1 8 49 107.3 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

44 H482/17 28 M 3 3.6 21 118.9 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

45 H542/17 50 F 1.5 6.8 35 72 PANCYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

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46 H575/17 46 F 1.4 9.5 25 88 PANCYTOPENIA NORMOCYTIC NORMOCHROMIC ANEMIA MYELO FIBROSIS

47 H593/17 36 M 2.9 4.8 97 94.3 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

48 H600/17 45 F 2.6 5.3 50 76.5 PANCYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MYELO FIBROSIS

49 H615/17 42 M 1.7 3.2 21 97.7 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

50 H623/17 37 F 3.5 3.7 32 127.6 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

51 H654/17 48 M 2.6 4.7 77 96.7 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

52 H660/17 75 M 3.3 3.9 98 120 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

53 H724/17 34 F 2.7 5.5 57 88.4 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

54 H728/17 40 F 11 7.5 47 85.3 BICYTOPENIA DIMORPHIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

55 H748/17 46 M 3.1 3.9 66 112 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

56 H752/17 52 F 1.6 5.2 40 98.7 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

57 H833/17 1 M 2.3 8 27 87.6 PANCYTOPENIA NORMOCYTIC NORMOCHROMIC ANEMIA METASTASIS

58 H940/17 31 M 5 5.9 79 97.6 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

59 H941/17 48 M 3.2 4.3 128 71.1 BICYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MYELODYSPLASTIC SYNDROME

60 H942/17 40 M 3.5 7.1 74 97.7 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

61 H943/17 42 F 9 9 90 80.3 BICYTOPENIA DIMORPHIC ANEMIA IMMUNE THROMBOCYTOPENIA

62 H1016/17 52 F 6.2 8.6 90 89 BICYTOPENIA DIMORPHIC ANEMIA ACUTE LEUKEMIA

63 H1028/17 32 M 2.2 7.6 17 121 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

64 H1065/17 75 M 5.4 4.7 73 79 BICYTOPENIA DIMORPHIC ANEMIA METASTASIS

65 H1165/17 16 F 4.4 9 35 83.8 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

66 H1177/17 27 F 3.2 3.2 36 114.7 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

67 H1228/17 60 F 4.7 5.4 76 109 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

68 H1231/17 48 M 2.9 4.4 61 112 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

69 H1241/17 39 M 4.1 5.7 92 88.2 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

70 H1242/17 28 M 0.6 5 52 121 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

71 H1243/17 27 M 2.9 3.9 80 93.1 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

72 H1303/17 55 F 1.9 4.7 95 116.9 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

73 H1312/17 60 F 2.6 6.7 47 95.6 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

74 H1322/17 82 F 2.4 5.2 78 119 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

75 H1323/17 32 F 1.3 5.2 3 109 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

76 H1336/17 47 F 2.2 6.4 99 89.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

77 H1394/17 62 F 1.8 3.5 10 87 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

78 H1450/17 40 M 3.5 6.6 34 92 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

79 H1454/17 62 M 4.2 6.6 50 75.6 BICYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

80 H1500/17 13 M 6.2 9 9 83.6 BICYTOPENIA DIMORPHIC ANEMIA IMMUNE THROMBOCYTOPENIA

81 H1526/17 52 M 2.8 9.6 40 91.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

82 H1559/17 70 F 3.5 7.2 98 128.9 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

83 H1655/17 22 F 1.7 6.6 252 77.9 BICYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

84 H1661/17 28 F 3.6 6.9 80 89.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

85 H1672/17 35 F 2 4.4 92 90 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

86 H1682/17 63 F 5 8.4 90 105.2 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

87 H1692/17 30 F 3 5.4 54 77.5 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

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88 H1699/17 28 F 5.5 5.2 16 116 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

89 H1713/17 18 F 6.5 6.9 95 75.9 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

90 H1717/17 30 M 1.8 4.7 36 98.5 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

91 H1812/17 62 M 7 5.5 88 112.3 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

92 H1819/17 65 M 8 7.5 34 105.4 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

93 H1847/17 45 F 8 5.4 55 79.3 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

94 H1885/17 35 F 6 5 99 94.6 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

95 H1892/17 29 M 3 4 70 88.4 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

96 H1913/17 49 M 4 5 70 98.3 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

97 H1/18 17 F 3.5 6.2 66 106.1 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

98 H230/18 5 M 8.6 10 21 79.6 BICYTOPENIA DIMORPHIC ANEMIA IMMUNE THROMBOCYTOPENIA

99 H231/18 21 M 3 5.3 68 110 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

100 H239/18 58 M 2 9 55 85.9 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

101 H245/18 4 M 3.8 6.9 8 77 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

102 H266/18 55 M 3.8 6 40 105.3 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

103 H271/18 32 M 6.7 6.5 88 81 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

104 H272/18 45 F 9 9 87 81.1 BICYTOPENIA DIMORPHIC ANEMIA PLASMACYTOMA

105 H350/18 18 F 1.4 8 40 81.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

106 H351/18 36 M 4.6 5 91 116 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

107 H355/18 39 M 2.8 7 150 111 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

108 H356/18 70 M 2 5.3 43 122 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

109 H363/18 21 M 1.2 7.5 54 87.7 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

110 H372/18 30 M 1.6 4.6 20 92 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

111 H472/18 30 M 1.8 8.4 55 111.9 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

112 H473/18 30 M 2.2 5.2 90 89.8 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

113 H490/18 44 M 3.8 6.6 76 121 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

114 H494/18 43 F 2 4.5 90 77.3 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

115 H516/18 45 M 6.3 6 84 90 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

116 H614/18 44 M 3.8 5 40 81.5 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

117 H663/18 34 F 2.6 7.5 85 94.4 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

118 H667/18 25 F 5 7.8 83 100.5 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

119 H713/18 24 F 3 5.5 51 119 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

120 H742/18 20 M 3.6 8.3 40 93.4 PANCYTOPENIA ACUTE LEUKEMIA ACUTE LEUKEMIA

121 H748/18 28 F 2.7 7.9 50 81.1 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

122 H781/18 28 M 1 6.6 11 96.4 PANCYTOPENIA SUBLEUKEMIC LEUKEMIA ACUTE LEUKEMIA

123 H793/18 40 F 1.8 5.2 26 91.9 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

124 H811/18 49 M 1.2 6.6 70 95.3 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

125 H816/18 75 M 3.8 5.2 41 103.3 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

126 H875/18 85 F 2.7 9 16 94.4 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

127 H882/18 47 F 1.6 5.9 37 98.5 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

128 H883/18 72 M 7.3 3.1 37 86.7 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

129 H930/18 9 F 10 9 34 68.9 BICYTOPENIA DIMORPHIC ANEMIA IMMUNE THROMBOCYTOPENIA

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130 H955/18 30 M 5 5.9 89 103.4 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

131 H959/18 74 F 3.1 7.4 26 74.8 PANCYTOPENIA DIMORPHIC ANEMIA MYELODYSPLASTIC SYNDROME

132 H963/18 32 M 3.4 4.4 59 101.6 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

133 H964/18 35 M 2.9 5.4 44 94.6 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

134 H973/18 48 F 3.6 8.7 87 82.1 PANCYTOPENIA NORMOCYTIC NORMOCHROMIC ANEMIA MYELODYSPLASTIC SYNDROME

135 H980/18 55 M 3.6 9 66 79.4 PANCYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

136 H981/18 45 M 7 8 26 93.8 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

137 H982/18 40 F 4.7 6.1 61 108.4 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

138 H1039/18 32 M 2.8 5 160 100.6 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

139 H1078/18 49 M 2.5 6.5 50 100.8 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

140 H1105/18 28 M 3.7 8.6 59 120 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

141 H1114/18 44 F 10 8.3 55 108.1 BICYTOPENIA ACUTE LEUKEMIA ACUTE LEUKEMIA

142 H1177/18 17 M 2.9 6 63 96.1 PANCYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

143 H1179/18 60 M 4.2 5.5 99 111.4 BICYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

144 H1184/18 52 F 2.6 6.3 43 86.1 PANCYTOPENIA MICROCYTIC HYPOCHROMIC ANEMIA MICRONORMOBLASTIC ERYTHROID HYPERPLASIA

145 H1200/18 45 M 3.4 8 86 101.8 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

146 H1208/18 38 F 5.3 6 66 106.9 BICYTOPENIA DIMORPHIC ANEMIA MYELODYSPLASTIC SYNDROME

147 H1273/18 48 M 10 8.8 62 91.2 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

148 H1282/18 20 F 7.8 7.1 62 79.1 BICYTOPENIA DIMORPHIC ANEMIA MYELODYSPLASTIC SYNDROME

149 H1283/18 65 M 2.7 6.8 31 107.6 PANCYTOPENIA MACROCYTIC ANEMIA MEGALOBLASTIC ANEMIA

150 H1294/18 45 M 4.5 5.6 67 95.2 BICYTOPENIA DIMORPHIC ANEMIA COMBINED DEFICIENCY

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II: ABBREVIATIONS

ANNEXURE II

ABBREVIATIONS

HSC HEMATOPOIETIC STEM CELLS

CFU COLONY FORMING UNIT

BFU BURST FORMING UNIT

MPP MULTI POTENT PROGENITOR CELLS

GMP GRANULOCYTE - MACRO PHAGE PROGENITOR

MEP MEGAKARYOCYTE - ERYTHROID PROGENITOR

PNH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

EPO ERYTHROPOIETIN

SCF STEM CELL FACTOR

MDS MYELODYSPLASTIC SYNDROME

HB HEMOGLOBIN

PLT PLATELET

MCV MEAN CORPUSCULAR VOLUME

IP IMMUNE THROMBOCYTOPENIA

III: PROFORMA

ANNEXURE III

PROFORMA

Name : IP NO :

Age : Ward :

Sex : Address :

Presenting complaints :

General examination :

Lymphadenopathy :

Organomegaly :

Provisional diagnosis :

Complete blood count :

Hemoglobin :

White blood cell count :

Platelet count :

Mean corpuscular volume :

Differential count :

Peripheral smear findings :

Bone marrow aspiration findings :