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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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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
1 1
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
1 1
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
1 1
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
1 1
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 :