Declaration by the candidate

1
Certify by the guide

2
Endorsement by the HOD, Dean of the institution

3
Acknowledgement

4
List of abbreviations used

5
Abstract

6
Contents

1. Introduction .................................................................................................................................. 9
2. Objectives ................................................................................................................................... 11
3. Review of Literature ................................................................................................................... 12
4. Methodology .............................................................................................................................. 47
5. Results......................................................................................................................................... 51
6. Discussion ................................................................................................................................... 54
7. Summary ................................................................................................................................... 118
8. Conclusion ................................................................................................................................ 119
9. Bibliography .............................................................................................................................. 120
10. Annexures ................................................................................................................................. 136
11. Proforma ................................................................................................................................... 137
12. Consent form ............................................................................................................................ 138
13. Master chart ............................................................................................................................. 139

7
List of Tables

List of Figures

List of Graphs

8
1. Introduction

There are three modalities to examine bone marrow, Bone marrow aspirate

cytology (BMA), bone marrow biopsy (BMB) and touch imprint cytology (BMI).

Bone marrow aspirate (BMA) gives cytological picture, bone marrow touch

imprints also gives cytological picture but cells are less in number and bone

marrow biopsy gives cytological as well as bone marrow architectural picture.

Only BMA is not sufficient enough to reach up to the diagnosis therefore in the

present study I did comparative study of the above three modalities for bone

marrow examination.

The technique of BMA has been universally accepted and widely used. BMB as

a diagnostic procedure is being increasingly used in recent years. Biopsy of the

bone marrow is an indispensable adjunct to the study of diseases of the blood

and may be the only way in which a correct diagnosis can be made.

If performed correctly, BMA is simple and safe; it can be repeated many times

and performed on outpatients. It seems to be safe in almost all circumstances,

even when thrombocytopenic purpura is present. However, when there is a

major disorder of coagulation, such as in hemophilia, it should never be

attempted without appropriate cover and checking by coagulation factor

assay prior to the procedure.

9
BMB is a little less simple, but it too can be performed on outpatients.

BMI is also a reliable diagnostic tool for determining the cellular composition.

The bone marrow evaluation may either confirm clinically suspected disease or

may provide previously unsuspected diagnosis. Although studies have

compared the role of BMA for diagnosing various hematological disorders but

fewer studies have compared the relative value of BMB and BMI.

The present study comprises of 30 BMA, BMB and BMI carried out in Dhiraj

General Hospital, Piparia, to compare relative amount of information obtained

in each.

10
2. Objectives

1. To compare the role of BMA, BMB and BMI in arriving at a diagnosis.

2. To assess diagnostic accuracy of the procedures.

3. To correlate the result with the other studies.

11
3. Review of Literature

HISTORICAL PERSPECTIVE:

The oldest known procedure carried out on mankind is that of trepanning. Skulls

8,000–10,000 years old showing evidence of medical intervention have been

found in Europe, Northern Africa, Asia, New Guinea, Tahiti and New Zealand (Fig

1). Many of these ‘patients’ survived is shown by evidence of healing of their

bones.(1)

In Peru, from where a large amount of information comes, the procedure is likely

to have been carried out to relieve headaches, mental illness and to relieve

intracranial pressure. Peruvians used sharp knives of obidian, stone and bronze

for trephination, as well as bone instruments, bandages, native cotton and other

auxiliary items.(2)

One of the most spectacular operations described by Celsus, the roman

physician, was trephination. He recommended it for removal of damaged

cranial bones and as a therapeutic measure for relieving headaches. For the

excision of small areas of bone Celsus described a specialized instrument, a

surgical modulus or crown trephine. (Fig 2)(3)

12
Crown trephines (Fig 3and 4) were used as late as the 1700s for therapeutic

measures but not for diagnostic use (4). Similar trephines have continued to be

used by surgeons to the present day for therapeutic purposes on the skull.(Fig 5)

Surgical trephine BMB is an older procedure than BMA and BMI. Pianesein

1903(1) was the first to obtain marrow from the epiphysis of the femur. Sternum,

iliac crest and tibia were used by the subsequent workers for BMB. He described

a case of anemia because of bone marrow infiltration by Leishmania as

‘Leishmania Infantum’. The technique was discarded because of difficulty in

obtaining adequate material and the laborious procedure for preparing

sections. Thus BMA continued to dominate in hematologicalpractice. (5)

Ghedini in 1908 (6) suggested trephination of the medial part of the epiphysis of

femur using a hematological spoon. However, he analyzed tibial aspirates after

tissue sections rather than marrow aspirates. His technique did not gain wide

acceptance.

Morris and Falconer in 1922 (7) introduced a method for tibial marrow biopsy

using a drill like instrument that produced a marrow specimen very similar to that

obtained today.

13
Seyfarth in 1922 (8) developed a puncture needle for open biopsy of the sternal

method at the point between 3rd and 4thribs. He obtained satisfactory smears,

touch preparations, wet preparations and blocks for sectioning. These

procedures were carried out without benefit of adequate anesthesia but gowns

and gloves were recommended.

In 1927, Anirkin,(9) a Russian physician, obtained bone marrow from the sternum

using a lumbar puncture needle. It is noteworthy that Anirkin worked at the

same Military Medical Academy of Leningrad as the famous histologist

Maximow, theillustrious psychologist Pavlov, and the last Tsar’s physician, Botykin,

who is attributed for the first description of viral hepatitis. Anirkin published the

results of 103 procedures in the Russian ‘news of surgery’ journal (Anirkin,

1929).(9) No complications were reported. Anirkin claimed that marrow

aspiration stimulated marrow activity! Anirkin’s technique was used not only for

haematological disorders but also for the recognition of typhus and tuberculosis.

Peabody (1927) (10) , carefully prepared sections of curetted tibial marrow from

patients with Pernicious anemia. Arjeff (1931)(11) introduced needles with a

guard and Grunke (1938)(12) still recommended a short lumbar puncture

needle for marrow aspiration with the help of a wooden malleton the sternum.

14
The report by Custer and Ahlfeldt (1932)(13) included an account of their

experience with biopsy of the sternal marrow. A disk of the ventral plate of 1 cm

diameter was removed with a trephine, an elaborate operating technique at

the time. This report also demonstrated the value of obtaining histological

sections and touch preparations of the marrow compared with examining

smears.

The needles developed by Klima and Rosegger (1935)(Fig 6) (14) have guards.

Leitner (16) further modified the needle with the guard running on a thread. (Fig

7)A modern Salah needle with a guard is shown in Fig 8. The later was probably

the commonest in use in the late 20th century.

Henning and Korth (1934)(15) suggested a cannula with a side opening to

facilitate irrigation of the marrow cavity. Their cannula was graduated in

centimeters. These workers stated that they could obtain bone marrow by

injecting 1 ml of heparin or sodium citrate solution even when simple

puncturehad failed. They rarely carried out marrow irrigation. Until 1939, many

authors felt local anesthetics was unnecessary (Leitner et al, 1949)(16). Between

1929 and 1938 bone marrow samples were taken from healthy volunteers.

Faber, Anirkin’s assistant (9), published the results of normal bone marrow.

15
The first deaths following sternal puncture were reported in 1943 and 1944; the

sternum was completely penetrated and the right side of the heart punctured. It

was then recommended that the sternal puncture needle be driven through the

outer plate of the bone by gentle taps from a small hammer (Whitby & Britton,

1946). (17)

Turkel and Bethell (1943)(18) described a micro trephine of about 2 mm bore,

which could be passed through a hollow introducing needle only slightly larger

than a marrow aspirating needle. Noskin incision was necessary and it could be

used on the sternum. The samples obtained were small and fragile.

Although the pelvis contains 50% of the body’s marrow, it was not until 1950 that

the pelvis was suggested as a source of a specimen (Rubinstein, 1950) (19). One

assumes that prior to this, either suitable needles were not available or the site

not considered. Bierman (1952)(20) first suggested using posterior iliac crest,

which is the preferred site for both aspiration and biopsy. The Sacker-Nordin

trephine (Sacker & Nordin, 1954)(21), which could safely be used on the iliac

crest, provided sufficient material for accurate diagnosis. Waterfield

modification iliac crest aspiration is shown inFig 9.

In response to a 5% failure rate in carrying out aspiration biopsies, McFarland

and Dameshek (1958)(22) described a technique for trephine biopsy using the

16
Vim-Silverman biopsy needle (Silverman, 1938)(23). These biopsies were carried

out in left lateral position from the right iliac crest using local anaesthetic and

without incision of the skin.

Ellis & Westerman (1964)(24) reported on 1445 cases using a modification of the

Vim-Silverman needle between 1959 and 1963. The modified needle contained

finger grips, an assembly stilette and an obturator that locked in position, in

addition tobeing larger and sturdier. These biopsies were often carried out in the

outpatient department, and 5% were unsatisfactory for analysis. The biopsy was

of specific diagnostic value in 11% of cases where an aspirate alone would not

have been sufficient. The trans-ilial wide bore needle was designed to

obtain‘transfixing’ or cortex to cortex iliac crest biopsies (25). A commonly used

trephine needle in the 1960s/1970s was the Gardner’s trephine needle with a

serrated end (Fig 10).

Jamshidi and Swaim in 1971(26) introduced BMB needle and made the

procedure simpler and less painful with better processing mode and improved

light microscopic techniques (Fig 11).

Biopsies taken with an electric drill were performed exclusively from the anterior

iliac crest (Burkhardt, 1971).(28)

17
Islam (1982)(29) described an improvement whereby sampling error was

reduced. The needle (Fig 12), designed to obtain marrow samples from the

posterior iliac crests, had 14 side holes in the distal portion of the needle. The

proximal end of the needle was fitted with a large metal bar allowing a firm grip

and smoother operation.

The modern biopsy needles (Figs 13 to 15) are very similar to those used in the

past. The main areas of progress have been in improving the success rate of

acquiring a satisfactory sample with, for example, a Traplok needle. This features

‘forceps’ that capture large undamaged samples. Local, intravenous

andgeneral anaesthesia has reduced the amount of pain suffered by the

patients undergoing biopsies, but improvements inadequate analgesia

continues (27). Marrow needles are rarely used for fluid administration. A modern

example is shown in Fig 13.Economical disposable needles will be increasingly

available as more suppliers compete for the market. Better accuracy in

reporting has made the acquisition of a good quality biopsy almost mandatory.

For the foreseeable future there is no prospect of trephine biopsies losing their

appeal.

Though well accepted, fracture of the core was noticed in few cases. Prof.

Burkhardt in 1982(28) pioneered the plastic embedding technique, but it

required the use of an ultra-microtome.

18
Gatter KC (1987)(1) is of the opinion that the paraffin technique is equally good

if a little care is taken for processing. Thus in last three decades, the improved

trephine design of the needle, improvement in biopsy technique and technical

progress in their preparations have provided additional impetus to the study of

bone marrow as an organ with its architecture and components intact in their

natural spatial context. This has offered a broader basis for comprehension of its

function in health and disease.

In 1988, a patent application was filed for a powered biopsy needle (30)that

had a replaceable and disposable needle. After market research it was felt

that, in view of the excellent non-mechanical biopsy needles available, it should

not be introduced.

NORMAL BONE MARROW STRUCTURE

Nucleated red cells were first observed in the bone marrow by Neumann

(1868)(31). He also discovered the transformation of fatty marrow into red

haemopoietic tissue in anaemia.

Schilling (1925)(32) showed the importance of the clinical examination of the

blood. A microscopic examination of bone tissue in a blood disorder was done

19
in 1846 by John Dalrymple (1804–1852) of Dublin on a patient with multiple

myeloma (33).

The value of morphological examinations of the blood was shown by Ehrlich

(1879)(34), who used appropriate staining methods to classify the various blood

and bone marrow cells.

Pappenheim (1898)(35) built on the findings of Ehrlich (1891) (34) by using

Romanowsky eosin and methylene blue, which permitted better cell

differentiation. Naegeli (1900) and Schilling (1925)(36) soon began to link up the

reactions of the peripheral blood with changes at the site of origin of the red

cells. Investigations at first depended on postmortem findings. By the 1940’s the

heterogeneity of even the lymphocytes wasrecognized. More recently,

haematological diagnoses have been much enhanced by the addition of flow

cytometry and molecular genetics.

Present knowledge of bone marrow is as follows:

Bone marrow is a mesenchyme-derived, soft, semisolid, red gelatinous

substance occupying the medullary cavities of the axial skeleton. In adults,

bone marrow is the primary site of effective hematopoiesis and is composed of

bone, vascular structures and hematopoietic tissue. The medullary cavity is

20
encased by trabecular bone consisting of periosteum, cortical and subcortical

bone. Arterioles, sinusoids, and small peripheral nerves traverse the interstitial

space. The medullary cavity contains hematopoietic cells, stromal cells and

extra-cellular matrix.(37,38)

Age of the patient must be taken into consideration while assessing bone

marrow cellularity which refers to relative amount of hematopoietic and fat

cells. In the first decade, the marrow cellularity is 79%, gradually decreasing to

around 29% by 8th decade.(37,39)

ERYTHROCYTES: Erythroid series of cells are in inter-trabecular location forming

islands, nodules or clumps. The immature cells are in the centre of the island,

while mature cells are in the periphery of the islands. Macrophages with

hemosiderin pigment are seen in the vicinity of the erythroid islands. All the

stages of maturation can be seen in the population. Normal myeloid-erythroid

ratio in the biopsy is 3-15:1.(41)

GRANULOCYTES: All the series of granulocytes and their precursors are

identifiable in the bone marrow biopsy. They are para-trabecular in location

with immature cells towards the trabecular bone and maturing cells towards the

marrow space. Neutrophilic (brownish, punctuate) and eosinophilic (larger and

yellowish red) granules are readily distinguished. Basophils are infrequently seen.

21
MEGAKARYOCYTES: They are para sinusoidal in location. These are the largest

cells seen in the bone marrow ranging from 12-150 microns and are highly

pleomorphic. Mature megakaryocytes show eosinophilic cytoplasm with

variable granularity. The nucleus is coarsely cerebriform and multilobated. The

platelets are directly shed into the sinusoids.

MONONUCLEAR MACROPHAGE SYSTEM:

MONOCYTE SERIES: Monoblasts are morphologically similar to myeloblasts

except that their nuclear shape may be slightly clefted or lobulated.

Promonocytes(15-20 microns) are larger than monoblasts with rounded nuclei

and one or more prominent nucleoli. Monocytes (15-18 microns) have

abundant greenish cytoplasm with intracytoplasmic vacuoles. The nucleus is

eccentric, kidney shaped with fine and lacy chromatin. They are easily

confused with granulocytic precursors.

Macrophages are derived from monocytes and function as phagocytic cells in

the bone marrow and other tissue sites. Macrophages are larger than

monocytes measuring 20-30 microns in diameter. The nucleus is large, round to

oval with lace-like chromatin and abundant pale blue vacuolated cytoplasm

containing azurophilic granules and intra-cytoplasmic inclusions which vary in

size and shape and contain phagocytosed debris. They are usually present in

22
 the centre of the erythroid islands, plasma cell islands, and adjacent to the

endothelial cells.

LYMPHOCYTES: The earliest morphologically identifiable cells are lymphoblasts

which are present in the inter-trabecular region. They have a high nuclear

cytoplasmic ratio with a narrow rim of deep blue cytoplasm and an oval

hyperchromatic nucleus with one or two nucleoli. Prolymphocytes have larger

amount of cytoplasm and larger nucleus with coarse chromatin. The mature

lymphocytes measure 7-10 microns in diameter and are characterized by a

round nucleus with coarse, condensed chromatin, inconspicuous nucleoli and

scanty deep blue cytoplasm.

Lymphoid aggregates have been described in four configurations.

 Nodules with germinal centers.

 Sharply demarcated nodules.

 Nodules with irregular borders.

 Small aggregates of lymphoid cells.

PLASMA CELLS: The characteristic location of plasma cells is along the adventitia

of small blood vessels but they can also be found singly and in groups all

through the intertrabecular location. They measure 10-18 microns and are

23
characterized by an eccentric nucleus with coarse chromatin and deeply

basophilic cytoplasm containing a perinuclear clear zone.

MARROW STROMA: Bone marrow stromal cells consist of adipocytes, osteoblasts,

osteoclasts, endothelial cells and fibroblast-like reticular cells. Adipocytes (fat

cells) are the largest cells in the bone marrow stroma, measuring approximately

80-90 microns in diameter. They lie in close contact with hematopoietic cells and

other stromal cells. Adipocytes are inversely proportional to the hematopoietic

cells. The mechanism of this inverse relationship between hematopoietic cells

and fat cells is not well established.

Osteoblasts are large, ovoid or cuboidal cells measuring 20-50 microns in

diameter. The osteoblasts have a small eccentric nucleus and an abundant

basophilic cytoplasm with a clear Golgi zone located away from nucleus.

Osteoblasts are rarely seen in normal adult bone marrow.

Osteoclasts are large, multinucleated cells with abundant pale blue cytoplasm

containing numerous azurophilic granules. They can measure 100 microns or

greater in diameter. The individual nuclei are separate, uniform and round.

Endothelial cells are elongated cells containing a flat nucleus with condensed

chromatin and a moderate amount of cytoplasm.

24
Reticular cells are group of cells that form a reticulum or syncytium. These cells

are associated with reticular fibers which they produce and which form a three-

dimensional supporting network that holds the vascular sinuses and

hematopoietic elements. The fibers can be visualized by light microscopy and

after silver staining.

In September 1974, Bearden JD et al. studied the diagnostic value of bone

marrow biopsy and bone marrow aspiration in neoplastic disease. The Jamshidi-

Swaim biopsy needle was utilized to perform 205 bone marrow biopsies,

accompanied by simultaneous bone marrow aspirates, on patients with

lymphoma, leukemia, and a variety of solid tumors. There was no significant

morbidity. There were 67 positive findings with biopsy and 42 with aspiration.

They found that the two techniques were complementary in Hodgkin's disease,

non-Hodgkin's lymphoma, breast carcinoma, bronchogenic carcinoma,

malignant melanoma, and in leukemia. They have examined the bone marrow

biopsies and aspirates with respect to the adequacy of the bone marrow biopsy

specimen, the number of positive biopsies in the various categories of neoplasia,

and the disparity of biopsy and aspirate. They found that 28 of the 67 positive

biopsies (41.8%) had negative aspirates. Both the procedures were

complementary to each other. They felt that for diagnostic purpose both the

procedures can be done simultaneously as BMA gives better morphology of the

25
cells and BMB gives a good picture regarding the pattern of distribution of cells.

They found that BMB was especially useful in diagnosis of Hodgkin’s disease,

tuberculous granulomas and metastasis of non-hematological malignancies.

They also found these procedures quite useful in cases where malignancies

were not suspected; BMA and BMB are very useful and still an important

diagnostic tool. While performing the BMA and BMB simultaneously, employment

of proper technique should be kept in mind so as to yield the maximum material

and reduce discomfort to the patient by not repeating the procedure due to

inadequate material.(42)

Percutaneous micro trephine bone marrow biopsy by the Jamshidi-Swaim

method was employed in the investigation of selected patients during a 1-year

period by Mills AE (1976). They confirmed the importance of this procedure as a

method of diagnosing bone marrow lesions characterized by altered

architecture or malignant infiltration in aplastic anaemia, myelofibrosis,

Hodgkin's and non-Hodgkin's lymphoma and carcinomatosis. They found that

bone marrow aspiration is frequently not helpful in the diagnosis of these

diseases. They stated that both aspirated and biopsy material should be

examined together, and the two methods are often complementary.(43)

26
Loannides K and Rywlin AM (1976) stated that bone marrow aspiration isuseful in

making out better individual cell morphology whereas biopsy is useful in bone

marrow architectural pattern and distribution.(44)

Brynes RK (1978) stated that a thorough bone marrow morphologic study

involves examination of peripheral blood smears, direct, particle, and buffy coat

bone marrow smears, trephine biopsy imprints, particle and trephine biopsy

sections, and marrow volumetric data. The information obtained from the study

of these various specimens is complementary. They stated that utilization of

biopsy material by the methods described provides complete, accurate and

reproducible information and minimizes the necessity for repeating a biopsy for

morphologic diagnosis or ancillary studies.(45)

Burkhardt R et al (March,1982) examined semi-thin sections of 8216 un

decalcified biopsies of patients with hematological disorders. They stated that

bone marrow biopsies are essential for the differential diagnosis of most

cytopenia and for the early recognition of fibrosis which most frequently

occurred as a consequence of megakaryocytic proliferation in the

myeloproliferative disorders. They found different patterns of bone marrow

involvement in the lymphoproliferative disorders and both their type and extent

constituted factors of prognostic significance. They concluded that bone

27
marrow biopsies provide indispensible information for the diagnostic evaluation

and the follow-up of patients with haematological disorders.(46)

Two hundred and eight serial bone marrow samples from 49 consecutively

diagnosed children with neuroblastoma were studied retrospectively for

evidence of tumour invasion by I M Franklin in 1983. Bone marrow involvement

was found in 24 patients at diagnosis and in four more at a later stage in their

disease. They found that trephine biopsies were more effective than aspirates

for tumour detection in 20%of the 154 paired aspirate/trephine procedures,

whilst the reverse was the case in 7%. Imprints of trephines gave no additional

information. Bilateral sampling (aspirates and trephines) improved the tumor

detection rate by 10% over that attained by sampling a single site. They found

some correlation between specific appearances in aspirate and in trephine

samples. Bilateral iliac crest bone marrow aspirates and trephine biopsies are

indicated in children with neuroblastoma, both for initial staging and for

monitoring of progress.(47)

Pasquale D at al (August 1986) compare Bone marrow aspirate particle smears,

biopsy imprints, and biopsy sections to determine the accuracy of the three

samples in assessing for overall cellularity, differential cell count, megakaryocyte

density, iron stores, and tumor infiltration. Aspirate particle smears and biopsy

28
imprints were stained by Wright-Giemsa method. Aspirate particle smears were

also stained with Prussian-blue. Biopsy sections were 1 1/2-2 micron thick and

were prepared from non-decalcified plastic embedded samples and stained

with combined Prussian-blue-hematoxylin-eosin, and Giemsa. One hundred-

eight sets of specimens from 99 patients were examined. In 20 cases, chi-square

analysis showed a comparable degree of cellularity (p less than 0.001) and

megakaryocyte density (p less than 0.001) among the three preparations.

Differential count comparison by regression analysis indicated that mean

percentages of neutrophilic cells in the proliferation compartment were

comparable in the three groups (p less than 0.01). A better correlation was

obtained among the three groups in the percent neutrophilic cells in the

maturation-storage compartment, normoblasts, eosinophils, and plasma cells (p

less than 0.001). Lymphocytes in the aspirate smears correlated with the biopsy

imprints (p less than 0.01) but not with the biopsy sections (p greater than 0.05).

Monocytes did not correlate in any of the groups (p greater than 0.05). In 47

cases, chi-square analysis of iron stores in the aspirate particle smears correlated

well with those in the biopsy sections (p less than 0.001). Fifty-two marrows that

were done for staging non hematological malignancies revealed malignant

cells in 21 cases, biopsy sections were positive in all, biopsy imprints were positive

in 19 (90%), and aspirate particle smears were positive in 7 (33%). Thirty-six

marrows done for staging non-Hodgkin's lymphoma showed involvement in 13

cases. Twelve (92%) biopsy sections, three (23%) biopsy imprints, and nine (69%)

29
aspirate particle smears contained lymphoma cells. They concluded that a

satisfactory evaluation of marrow samples for diagnostic studies can be

achieved by examination of biopsy sections along with aspirate particle smears

or biopsy imprints. Any of the three marrow preparations alone is not sufficient

for accurate diagnosis in all cases. The biopsy imprint is an accurate modality for

identifying non hematological tumor metastasis in the bone marrow.(48)

Bone marrow cellularity estimated by biopsy was compared to the cellularity of

the aspirate particle smear and the volumetric method in two groups of children

(Ozkaynak MF, Scribano P, Gomperts E, et al,nov 1988). In the first group, 101

consecutive bone marrow biopsies and aspirates were evaluated from patients

with various diagnoses. In the second group, 20 patients with acute non

lymphoblastic leukemia were studied with 80 biopsies and aspirates at diagnosis

and following chemotherapy. A wide discrepancy was noted between bone

marrow cellularity confirmed by biopsy vs. the particle smear or the volumetric

method in both groups. They found that neither the volumetric nor the particle

method provides a good correlation of bone marrow cellularity. They also

compared the volumetric method with that of the biopsy to evaluate the

efficacy of the former method in detecting bone marrow infiltration by solid

tumors. They found that the volumetric method is an accurate modality of

identifying solid tumor infiltration in the bone marrow.(49)

30
Sabharwal BD at al (October 1990) evaluated bone marrow aspirate particle

smears, imprints and biopsy sections in 30 cases. They concluded that core

needle biopsy of the bone marrow is a valuable diagnostic aid for measurement

of marrow cellularity, metastatic tumors and fibrosis. It should not be taken as a

substitute for examination of the marrow by aspiration smear but is a

complementary procedure which affords several advantages. Bone marrow

biopsy was of maximum utility in myelofibrosis which was diagnosed on biopsy

alone. There were three additional cases with normal bone marrow aspiration in

which specific diagnosis could only be made from bone marrow biopsy

sections. New methodologies i.e. plastic embedding and semi thin sections of

un-decalcified bone marrow, to improve the cytological details of tissue

obtained by biopsy. Imprint preparations obtained from biopsy can be useful in

patients of malignancy but they found them to be of limited value except in

cases of dry tap.(50)

Varma N et al (1993) studied the relative efficacy of trephine sections, trephine

imprints and aspiration smears in yielding diagnostic and additional information

in 767 sets of bone marrow samples. Trephine sections were diagnostic in

significantly more cases as compared to trephine imprints and aspiration smears

(P < 0.001). Additional information was obtained in 326 trephine sections which

was not available from trephine imprints and aspiration smears. Significantly

more number of trephine sections provided diagnosis in case of dry tap/scanty

31
material, for assessment of lymphoma-tumor infiltration, cellularity, Perl's

reaction, megakaryocyte density and proliferating cell lines in myeloproliferative

disorders. Fibrosis of bone marrow, pattern of bone marrow involvement and

topographical alterations were appreciable only on trephine sections. The

differential counts done on trephine imprints and aspiration smears correlated

well and cyto-morphological characterization of immature cells (blasts and

promyelocytes) could be done on these two preparations. Although trephine

sections provide maximum information, all three preparations were found

complementing each other and should be evaluated simultaneously for

complete bone marrow interpretation.(51)

Hodges A, Koury MJ et al(1996 Nov-Dec) reviewed the procedures required to

perform and evaluate needle aspiration and biopsy .They concluded that

several tests require special handling when obtaining and processing bone

marrow samples. Serial bone marrow aspiration and biopsy studies can help in

the management of some bone marrow diseases. They state that current

procedures for obtaining and procuring bone marrow needle aspirates and

biopsies require close interaction between the clinical laboratory scientist (CLS)

and the physician. Multiple specialized assays require special handling at the

time marrow samples are obtained. They stated that serial bone marrow needle

aspirates and biopsies are very useful in guiding the clinical care of certain

patients. (52)

32
Aboul-Nasr R et al (1999) compared the differential counts of normal and

abnormal bone marrow from touch imprints with those from aspirate smears to

determine whether the touch imprint was reliable for independent routine use in

the examination of bone marrow and the classification of hematologic

abnormalities. Normocellular bone marrow specimens were obtained from 87

patients without hematologic abnormality. Abnormal bone marrow specimens

were obtained from 173 patients with treated or untreated neoplastic

hematologic disease, including acute myeloid leukemia, myelodysplastic

syndrome, chronic lymphocytic leukemia, non-Hodgkin lymphoma, hairy cell

leukemia, myeloma, and acute lymphoblastic leukemia. They found no

diagnostic difference in the differential counts from touch imprints and aspirate

smears of normo-cellular bone marrow, and although they found some

difference between the differential counts in certain cases of diseased bone

marrow, the touch imprint proved to be a reliable diagnostic tool for

determining the cellular composition of normal bone marrow and more reliable

for the diagnosis of bone marrow involved by a neoplastic hematologic disease.

Their findings suggest that evaluating touch imprints should be considered a

standard practice in examining bone marrow.(53)

In a study by Kini J et al(2001), 84% patients had hyper cellular marrow. 68% of

the patients had trilineage dysplasia. Bone marrow biopsy is mandatory in MDS

as it shows dysplastic maturation in all three elements. (54)

33
Fauci AS et al (2001) stated, if hypoplasia or aplasia is suspected, a bone

marrow biopsy should be performed to exclude MDS, Malignant lymphomas,

Acuteleukemias, Hairy Cell Leukaemia, Myelofibrosis and metastatic

carcinomas.(55)

Nanda A at al, july 2002, performed bone marrow aspiration and bilateral

trephine biopsies in 420 consecutive cases to determine the relative efficacy of

bone marrow aspiration as compared to that of trephine biopsy. The diagnosis

and findings made on bone marrow aspiration were compared with that made

on trephine biopsy in each case. Aspiration alone was sufficient in making a

diagnosis in 372 (88.6%) cases as it correlated well with the diagnosis made on

trephine sections. In the remaining 48 (11.4%) cases trephine biopsy was

necessary for making a diagnosis due to incomplete information provided by

aspiration or its inability to give a correct diagnosis. These cases were mostly

hypoplastic /aplastic marrow, myelofibrosis and marrow involvement by

metastatic tumor and lymphomatous infiltration. Often a bilateral marrow biopsy

picked up the diagnostic lesion. They concluded that the decision to perform a

marrow aspiration alone or in combination with marrow biopsy depends on the

diagnosis being considered. In nutritional anaemia, most hematologic

malignancies and immune thrombocytopenias, marrow aspiration alone is

sufficient, but for detection of disorders with focal marrow involvement bilateral

marrow biopsies are a must.(56)

34
Pampa Ch. Toi et al (2007) in a retrospective study reviewed in 160 cases where

BMA and BMB results are correlated with the clinical history. The advantage of

each method was analyzed. They found that 61.25%of the cases showed a

positive correlation between bone marrow aspiration and bone marrow biopsy.

However, they also found that tuberculous granulomas and Hodgkin disease

involvement of the marrow were detected better in bone marrow biopsies. The

advantage of both the procedures done together provided more material and

enabled us to study the cytomorphology of the cells, with the pattern of

distribution of the cells depending on the cases. However, when both the

procedures were done simultaneously, a proper technique is required so as to

yield good diagnostic material.(57)

Riley RS, Hogan TF, Pavot DR, et al (2004) reported on A pathologist's perspective

on bone marrow aspiration and biopsy. They found that bone marrow aspirate

and biopsy is an important medical procedure for the diagnosis of hematologic

malignancies and other diseases, and for the follow-up evaluation of patients

undergoing chemotherapy, bone marrow transplantation, and other forms of

medical therapy. They state that the recent development of bone marrow

biopsy needles with specially sharpened cutting edges and core-securing

devices has reduced the discomfort of the procedure and improved the quality

of the specimens obtained.(58)

35
Moid F et al.(april 2005) compared the relative value of aspirates and trephine

biopsies in the diagnosis of solid tumor metastasis and Hodgkin lymphoma, Sixty-

six cases where both aspirate and trephine biopsy were evaluated, there was a

22% positive correlation in the findings on aspirates and trephine biopsies. The

correlation between aspirates and trephine biopsies was highest in cases of

small cell carcinoma of the lung (3/11, or 36.3%) followed by breast carcinoma

(7/20, or 35%), prostate carcinoma (1/9, or 11.1%), and Hodgkin lymphoma

(1/20, or 5%). Two of five cases from the miscellaneous category demonstrated

simultaneous involvement of aspirate and trephine biopsy by a gastric

carcinoma as well as an adrenal gland carcinoma. They concluded that bone

marrow aspirate has only a minimal role, if any, in detecting bone marrow

involvement by Hodgkin lymphoma. In cases of breast carcinoma, small cell

carcinoma of lung, and prostate carcinoma, aspirate evaluation may confirm

trephine biopsy results or, more rarely, provide the sole confirmation of the

malignancy.(59).

The diagnostic impact of bone marrow cytology in combination with flow

cytometry analysis of aspirate smears and bone marrow histology together with

immune histochemical examination of trephine biopsies was compared in 141

routine cases by B. Lukas Grafa in 2005. Diagnoses achieved by the two

methods were concordant in 80.5% of cases. In discordant cases, clinical follow-

up data of at least one year confirmed the correctness of cytological and

36
histological diagnoses. For infiltration by malignant disease, both methods were

concordant in 86.5%of samples and correlated well for the degree of infiltration

(r = 0.64, p <0.001). Overall, regression analysis showed a good correlation for

cellularity(r = 0.67) lymphopoiesis (r = 0.75), granulopoiesis(r = 0.73) and

megakaryopoiesis (r = 0.65) while erythropoiesis displayed a lower degree of

correlation(r = 0.43, all p <0.001). Regression analysis on all immunological data

obtained by flow cytometry (FC) and immunohistochemistry (IHC) showed a

good overall linear correlation (r = 0.67,p <0.001), but significant differences

were found for a few phenotypic markers. Furthermore, the correlation was

found to be dependent on IgG subclasses and the fluorochromes used for FC.

Thus, analyses with IgG2 antibodies and phycoerythrin (PE) as fluorochrome

showed significantly more expression than IHC. They concluded that, cytology

and histology, both in associations with the respective immunopheno typing, are

of equal value in bone marrow diagnostics and should be used in combination.

However, in some specific settings, one of the two procedures might be

preferable.(60)

To compare the relative value of aspirates and trephine biopsies in the diagnosis

of solid tumor metastasis and Hodgkin lymphoma, Farah Moid (2005) et al

studied sixty-six cases showing bone marrow involvement by solid tumor and

Hodgkin lymphoma in bone marrow aspirates, bone marrow trephine biopsies,

or both. The diagnosis and findings made on aspirates were compared with

37
those made on trephine biopsies in each case. In those cases where both

aspirate and trephine biopsies were available for evaluation, there was a 22%

positive correlation in the findings on aspirates and trephine biopsies. The

correlation between aspirates and trephine biopsies was highest in cases of

small cell carcinoma of the lung (3/11, or 36.3%) followed by breast carcinoma

(7/20, or 35%), prostate carcinoma (1/9, or11.1%), and Hodgkin lymphoma (1/20,

or 5%). Two of five cases from the miscellaneous category demonstrated

simultaneous involvement of aspirate and trephine biopsy by a gastric

carcinoma as well as an adrenal gland carcinoma. They concluded that bone

marrow aspirate and bone marrow trephine biopsy should both be performed in

patients with proven or suspected malignancies where staging may affect

management. However, bone marrow aspirate has only a minimal role, if any, in

detecting bone marrow involvement by Hodgkin lymphoma. In cases of breast

carcinoma, small cell carcinoma of lung, and prostate carcinoma, aspirate

evaluation may confirm trephine biopsy results or, more rarely, provide the sole

confirmation of the malignancy.(61)

Sitalakshmi S, et al. (2005 Apr) observed that trephine biopsy is invaluable in

cases where the aspirate fails or is a dry tap as in the case of myelofibrosis, focal

marrow involvement as in granulomatous lesions, metastatic tumor and

lymphomas. In their study found that acute leukemia were diagnostic on

aspiration alone and trephine biopsy provided additional useful information.(62)

38
Lu XG, Huang LS, Xu XH, et al, In 2006, studied BMI to evaluate cellularity of

trephine biopsy from 272 patient. The imprints ware stained by Wright-Giemsa

method, and the bone marrow smears and imprints were examined

simultaneously according to the bone marrow cellularity criteria. They found that

in bone marrow cellularity, four grades (distinct decrease, extreme decrease,

distinct increase, and extreme increase) were significantly higher in bone

marrow imprints than those in bone marrow smears (P <0.05), but there was no

significant difference between bone marrow imprints and sections (P >0.05).

They concluded that to evaluate bone marrow cellularity, bone marrow imprint

is better than bone marrow smear. The combination of the two examinations

can make the diagnosis more convenient and quicker.(63)

Gupte R et al studied the role of trephine biopsy in immune compromised host,

especially HIV-positive patients, has been well studied in October 2008. They

attempted to evaluate the utility of marrow aspirate vis-à-vis trephine biopsy in

establishing a diagnosis in cases of pyrexia of unknown origin in immune

competent individuals, along with an analysis of haematological alterations in

these patients. Over a period of 8 years, 121 patients with pyrexia of unknown

origin underwent both bone marrow aspiration and trephine biopsy as a part of

diagnostic work-up. These cases were reviewed for their clinical data and

hematological findings, including detailed morphological features in aspiration

smears and trephine biopsies. Bone marrow aspiration and biopsy were

39
compared for their diagnostic efficacy in these patients. A wide age range (2-

65 years) was noted with a slight male predominance (2:1). Anemia was the

most common feature in peripheral blood findings, seen in 97.5% of patients.

Bone marrow aspiration was diagnostic in only 16.5% of cases, which revealed

leishmaniasis or pure red cell aplasia. Granulomas were infrequent in marrow

aspiration smears, as only two cases (1.6%) showed ill-defined epithelioid cell

collections. Compared to this, trephine biopsy offered a diagnosis in 76% of the

cases. Granulomas were a frequent finding in the trephine biopsy, being present

in 70% of the cases included. These cases diagnosed on biopsy (over those

diagnosed with aspiration smears) included lymphoma, tuberculosis, fungal

infection, sarcoidosis and hypocellular marrow. They concluded bone marrow

trephine biopsy is an important adjunct to aspiration in arriving at an etiological

diagnosis of patient with long-duration febrile illness, and should be routinely

performed in such cases. The presence of granulomas in trephine biopsy

increases the likelihood of an etiologic diagnosis in these patients.(64)

Xubo G et al, Oct 2009 , to better realize the features of peripheral blood (PB),

bone marrow (BM) aspirate and especially BM trephine biopsy in atypical

chronic myeloid leukemia (aCML) studied PB, BM smears in 35 cases of aCML

and compared with 84 cases of chronic granulocytic leukemia chronic phase

(CGL-CP), 39 cases of chronic myelomonocytic leukemia (CMML). In addition,

40
they evaluated characteristics of BM trephine biopsies in 21 cases of aCML and

compared with 68 cases of CGL-CP. They found that values of monocytes,

eosinophils, basophils, percentage of immature granulocytes and monocytes

(0.63 +/- 0.41 x 10(9)/L, 0.18 +/- 0.16 x 10(9)/L, 0.09 +/-0.08 x 10(9)/L, 6.27 +/-

3.09%, and 2.46 +/- 1.75%, respectively) useful in distinguishing aCML from CGL-

CP and CMML groups. The BM smears showed that striking dysgranulopoieis

(100%), dyserythropoiesis (48.6%), percentage of blasts, nucleated erythrocytes,

monocytes, eosinophils, and basophils (2.45 +/- 2.06%, 7.76 +/- 2.89%, 1.30 +/-

1.21%, 1.47 +/- 1.60%, and 1.15 +/- 1.08%, respectively) were all important

parameters for a diagnosis of aCML. On BM trephine sections, aCML was

characterized as hypercellularity, a moderate degree of reticulin fibrosis (71.4%),

lymphocytopenia (76.2%), plasmacytopenia (90.5%), abnormal localization of

immature precursors (28.5%), and absence of eosinophilia, basophilia,

monocytosis. They found that, BM imprints, immunohistochemical, and

cytochemical staining findings provide important morphological reference to

BM trephine sections and made the identification of nucleated cells more

convenient. They state that besides the findings observed in PB and BM aspirate,

features of BM trephine biopsy (including BM trephine section, BM imprint,

immunohistochemical, and cytochemical staining) can also aid in the diagnosis

of aCML.(65)

41
A study was conducted in October 2010 by Chandra et al, to analyze

hematological indicators which could predict marrow metastasis along with the

comparison of bone marrow aspirate, touch imprint and trephine biopsy, to

define an effective method for its early diagnosis. The study showed that there

was statistical significant difference in mean of mean platelet volume (MPV)

and platelet distribution width (PDW) between the cases and controls (P<0·001)

and MPV at cut off of <8 fl showed significantly high positive predictive value

(100%) and likelihood ratio (21·170) for bone marrow metastasis. Furthermore,

bone marrow imprint cytology detected metastatic cells in 96% of cases. Thus,

the study concluded that low MPV could be used as a probable indicator for

bone marrow metastasis and the meticulously prepared touch imprint smears,

along with bone marrow aspirate, provide an efficient method for rapid

diagnosis of metastasis.(66)

A retrospective study was done by Stifter et al in 2010 to compare the

percentage of plasma cells obtained by both methods of 59 MM patients. The

conventional differential count was determined in BMA to estimate the

percentage and cytologic grade of plasma cells. The pattern of neoplastic

infiltration and percentage of plasma cells were estimated on CD138 immuno

stained BMB slides microscopically and by computer-assisted image analysis

(CIA).Significantly higher values of plasma cell infiltrates were observed in

pathologist (47.7 ± 24.8) and CIA (44.1 ± 30.6) reports in comparison with

42
cytologist analysis (30.6 ± 17.1; P < 0.001 and P < 0.0048, respectively). BMB

assessment by pathologist counting and using CIA showed strongest correlation

(r = 0.8; P < 0.0001). Correlation was also observed between the pathologist and

cytologist counts (r = 0.321; P = 0.015) as well as comparing the percentage of

plasma cells in BMA and CIA (r = 0.27; P = 0.05). Patients with clinical stage I/II

had a significantly lower CIA plasma cell count than those with clinical stage III

(P = 0.008). Overall survival was shorter in patients with more than 25% of atypical

plasma cell morphology estimated in BMA (P = 0.05) and a higher percentage

of tumor cell infiltrates estimated by the pathologist and CIA (P = 0.0341 and P =

0.013, respectively). They suggested the combined analyses to be useful as a

routine procedure to achieve more accurate and informative diagnostic

data.(67).

A retrospective study was conducted to compare the role of bone marrow

aspirate, touch imprint and trephine biopsy to formulate an effective and rapid

method for diagnosing wide spectrum of hematological diseases by Smita

Chandra et al (2011). The study included total 565 cases of bone marrow

examination from January 2006 till May 2010. All the smears and sections were

reviewed for morphological details and findings on aspirate, imprint and biopsy

were compared to each other. The diagnostic accuracy of bone marrow

aspirate was 77.5%, imprint cytology 83.7% and that of biopsy was of 99.2%. The

study showed 78% positive correlation between aspirate and biopsy and 84.3%

43
between imprint and biopsy; 93.3% cases of metastatic solid tumors were

correctly diagnosed on imprint while only 70% cases were diagnosed on

aspirate cytology. They concluded that all the three preparations of aspirate,

imprint and biopsy complement each other. The assessment of iron status by

Perl’s stain is most suitable on aspirate smears but trephine biopsy remains the

gold standard for diagnosing granulomatous inflammation and hypoplastic/

aplastic anemia. Meticulously prepared imprint smears not only provide

cellularcomposition of marrow but may also be helpful in defining the

architecture of marrow especially in cases of metastatic solid tumors. Imprint

cytology smears should be standard practice for evaluating any marrow.(68)

Wilkins BS, May 2011 studied to avoide errors in the histological interpretation of

bone marrow trephine biopsy specimens requires an unprecedented degree of

collaboration between histopathologists, haematologists, specimen requesters,

specimen takers, laboratory technical staff and other scientific staff. A specimen

of good quality, with full, relevant clinical information is the essential starting

point. This must then be processed optimally and investigated appropriately,

involving immunophenotyping and molecular testing when needed. A wide

range of pathologies may involve bone marrow haemopoietic and stromal

components, and a systematic approach to analysing each of the components

in turn is required to avoid overlooking abnormalities; correlation with bone

marrow cells aspirated in parallel is particularly important. Final interpretation

44
should be a synthesis of the histological findings with information from such

haematological and other investigations, interpreted with due regard to clinical

context.(69)

Gong X, Lu X, Wu X, et al(Apr 2012) explored the role of imprints in routine bone

marrow (BM) diagnosis.The cellularity and diagnostic accuracy of BM imprints,

aspirate smears and trephine biopsy sections from 3781 patients were assessed

using routine cytochemical staining. Seventy-nine cases of lymphoma and 114

cases of plasma cell myeloma (PCM) were selected for correlation analysis of

tumor cell infiltration patterns. Another 21 cases of lymphoma were selected to

detect t(14;18)(q32;q21) and t(11;14)(q13;q32) by fluorescent in situ hybridization

(FISH) on BM imprints, and the G-banding technique was performed for

comparison. They found BM imprints were better than smears for evaluating

cellularity. In the BM imprint group, diagnostic accuracy for metastatic

carcinoma, myeloproliferative neoplasm, myelodysplastic/myeloproliferative

neoplasm and PCM was better than in the smear group, while accuracy for

megaloblastic anaemia, acute myeloid leukaemia, refractory cytopenia with

unilineage or multilineage dysplasia, refractory anaemia with excess blasts and

lymphoplasmacytic lymphoma was higher than in the section group, but not

statistically different from the smear group. They found good correlation of

infiltration patterns of lymphoma and myeloma cells was found between BM

imprints and sections (r=0.90 and 0.78, respectively). Detection of

45
t(11;14)(q13;q32) by FISH on imprints was higher than G-banding analysis. BM

imprints showed features of both smears and trephine sections. They found that

imprints superior to smears for evaluation of cellularity, and also better than

sections for analysis of cytological changes. In addition, they state that FISH on

BM imprints markedly improves the identification of chromosomal

abnormalities.(70)

46
4. Methodology

The present study consists of thirty cases where BMA, BMI and BMB were

performed at Dhiraj General hospital, Piparia from January 2013 to December

2013.

Complete clinical data were recorded including physical examination,

complete haematological study along with other relevant investigations and

proforma filled.

Following inclusion and exclusion criteria were adopted in this study:

Inclusion criteria:

Indications for bone marrow examination with due informed consent of patients

admitted to or attending OPD in Dhiraj General Hospital (41).

Indications for BMA:

1. Red cells disorders (e.g. Pancytopenia, Pure red cellaplasia )

2. Leukocytic Disorders(e.g. Subleukemia and aleukemic leukemia, acute

leukemia )

3. Megakaryocytic and platelets disorders(e.g. Unexplained thrombocytopenia

and thrombocytosis)

4. Myeloproliferative disorders

5. Myelodysplastic syndromes

6. Paraproteininemias

7. Pyrexia of unknown origin

47
8. Suspected lysosomal or other storage disorders

9. Iron store assessment

10. Metastasis

11. Unexplained hepatomegaly and/or splenomegaly

To correlate information obtained on BMA with BMI and BMB and to evaluate

the necessity for BMB and BMI, bone marrow aspirate and bone marrow biopsy

were performed in all cases.

CRITERIA FOR DIAGNOSIS OF PANCYTOPENIA:

Sr.No. Parameters Values

1. Haemoglobin <10.0 gm/dl

2. Total WBC Count <3500/µl

3. Platelet Count <1,00,000/µl

IN THE PRESENT STUDY ANEMIA WAS ARBITRARILY GRADED AS BELOW.

Sr.No. Grade Haemoglobin gm/dl

1. Normal >12.5

2. Mild anaemia 10-12.5

3. Moderate anaemia 7-10

4. Severe anaemia <7

48
 PROCEDURE:

1. BMA and BMB were performed on the Posterior superior iliac spine in all cases.

2. The patients were explained about the procedure and made to lie either in the

left or right lateral position with the knees drawn up.

3. The skin in the area of procedure (posterior iliac spine) was cleaned with 70%

ethanol and betadine (painting and draping). The skin, subcutaneous tissue and

periosteum were infiltrated with 5 ml of 2% lignocaine as local anaesthetic.

4. A small thin incision less than 0.5 cm was made. With a boring movement, the

Jamshidi needle was passed perpendicularly into the cavity of the bone. The

stilette was then removed and 0.2-0.3ml of marrow content was sucked with the

help of 10 ml syringe. Smears of BMA were made which was then fixed with

methanoland stained with Leishman-Giemsa stain.

5. After aspiration the same Jamshidi needle was re-introduced into the same site,

but on different tract with to and fro rotation to obtain core BMB tissue.

6. Whenever a trephine BMB core was obtained, imprints were taken before the

specimen was transferred into fixative. The bony core was gently rolled across a

slide, which was then fixed and stained.

7. The BMB specimen was fixed in 10% formalin overnight. After routine processing

and paraffin embedding, Haematoxylin and Eosin sections were studied.

PRODEDURE FOR HAEMATOXYLIN AND EOSIN STAIN:

o Slides were placed containing paraffin sections in a slide holder (glass or metal)

o Deparaffinized and rehydrated sections

49
o Dipped slides into a jar containing Haematoxylin stain for 15 minutes

o Rinse deionized water to allow stain to develop

o Dipped in Acid ethanol (to destain)

o Rinse with Tap water

o Blotted excess water from slide holder before going into eosin.

o Dipped slides into eosin containing jar for 5 minutes

o Dipped into Xylene jar

o For mounting a drop of DPX was placed on the slide using a glass rod, taking

care to leave no bubbles.

o Angle the coverslip and let it fall gently onto the slide. Allowed the Permount to

spread beneath the coverslip , covering all the tissue.

PROCEDURE FOR LEISHMAN - GIEMSA STAIN:

o BMA and BMI slides were fixed with methanol.

o After drying them, slides were kept on staining stand

o Leishman stain was poured on slides and kept for 5 minutes

o After washing slides with running tap water,again kept on stand and poured

Giemsa stain on each for 3-5 minutes.

o Deionized water was poured to prevent drying in last two steps.

o Washed with tap water, dried and properly labeled.

50
5. Results

The present study was conducted on patients admitted at Dhiraj General

Hospital, Piparia from January 2013 to December 2013. A comparative

evaluation of BMA, BMI and BMB was done on 51 cases. Based on their

hematological findings and other relevant investigations, the cases were

broadly classified into 11 groups listed in Table.1

TABLE.1: THE BREAKUP OF DIAGNOSIS OF 51 CASES

SR.N DIAGNOSIS NO OF CASES PERCENTAGES

O OF CASES(%)

1 MEGALOBLASTIC ANEMIA 10 19.6%

2 APLASTIC/HYPOPLASTIC ANEMIA 7 13.7%

3 IDA/MICRONORMOBLASTIC ERYTHROID 1 2.0%

HYPERPLASIA

4 DIMORPHIC ANEMIA 3 5.9%

5 ITP 1 2.0%

6 PLASMA CELL LEUKEMIA 2 3.9%

7 MPD 2 3.9%

8 LEUKEMIA 8 15.7%

9 NORMOCELLULAR MARROW 7 13.7%

10 E/O METASTASIS 8 15.7%

11 OTHER 2 3.9%

51
 TOTAL 51 100

MEGALOBLASTIC ANEMIA
3.9%
APLASTIC/HYPOPLASTIC
ANEMIA
IDA/MICRONORMOBLASTIC
19.6% ERYTHROID HYPERPLASIA
15.7%
DIMORPHIC ANEMIA

ITP

PLASMA CELL LEUKEMIA

13.7% 13.7%
MPD

LEUKEMIA

5.9% NORMOCELLULAR MARROW

15.7% 2.0%
E/O METASTASIS
2.0%
3.9% 3.9% OTHER

In the present study megaloblastic anemia [10 cases] was the commonest

hematological disorder encountered, followed by leukemia [8 cases].

The age distribution of different hematological disorders in the 51 cases is shown

in Table.2

TABLE.3: AGE DISTRIBUTION OF DIFFERENT HEMATOLOGICAL DISEASES IN 51 CASES

SR.N DIAGNOSIS AGE (YEAR)

< 10 11 to 21 31 41 >5

52
 20 to to to 1

30 40 50

1 MEGALOBLASTIC ANEMIA 4 1 1 2 2

2 APLASTIC/HYPOPLASTIC ANEMIA 1 2 2 2

3 IDA/MICRONORMOBLASTIC 1

ERYTHROID HYPERPLASIA

4 DIMORPHIC ANEMIA 1 1 1

5 ITP 1

6 PLASMA CELL LEUKEMIA 2

7 MPD 1 1

8 LEUKEMIA 4 3 1

9 NORMOCELLULAR MARROW 2 2 1 1 1

10 E/O METASTASIS 1 1 2 1 3

11 OTHER 1 1

TOTAL 13 9 7 7 1 14

53
 12
10
8
6 >51
4 41 to 50
2 31 to 40
0 21 to 30
11 to 20
< 10

Maximum number of cases were found in the age of >51 years [15 cases

(27.45%)] followed by less than 10 years [ 13 cases(25.49%]

The sex wise distribution of different diseases in 51 cases is shown in Table.3.

TABLE.3: SEX WISE DISTRIBUTION OF DIFFERENT HEMATOLOGICAL

DISEASES IN 51 CASES

SR.NO DIAGNOSIS MALE FEMALE

1 MEGALOBLASTIC ANEMIA 8 2

2 APLASTIC/HYPOPLASTIC ANEMIA 6 1

3 IDA/MICRONORMOBLASTIC ERYTHROID 0 1

HYPERPLASIA

4 DIMORPHIC ANEMIA 1 2

5 ITP 0 1

6 PLASMA CELL LEUKEMIA 1 1

54
7 MPD 1 1

8 LEUKEMIA 5 3

9 NORMOCELLULAR MARROW 7 0

10 E/O METASTASIS 3 5

11 OTHER 2 0

TOTAL 34 17

8
7
6
5
4
3
2
1 MALE
0
FEMALE

In present study male to female ratio in 51 cases with various hematological

disorders is 2:1.

The clinical presentation of the 51 cases with various hematological disorders is

shown in Table.4 and 5.

TABLE.4: CLINICAL PRESENTATION OF DIFFERENT HEMATOLOGICAL DISEASES IN 51

CASES

55
SR.N DIAGNOSIS CLINICAL FINDINGS AND PHYSICAL

O EXAMINATION

LYMPHADENOPA
HEPATOMEGALY
SPLENOMEGALY
H/O BLEEDING
BONE PAIN

JAUNDICE
STOOL OB
FEVER

THY
1 MEGALOBLASTIC 6 2 6 3 1 2

ANEMIA

2 APLASTIC/HYPOPLASTI 5 2 4 3 1 3

C ANEMIA

3 IDA/MICRONORMOBL 1 1

ASTIC ERYTHROID

HYPERPLASIA

4 DIMORPHIC ANEMIA 1 1 3 1 1

5 ITP 1

6 PLASMA CELL 2 2 2

LEUKEMIA

7 MPD 1 1 1 2 1 1

8 LEUKEMIA 6 4 4 7 3 3 5

9 NORMOCELLULAR 6 0 1 1 1 1 2

MARROW

10 E/O METASTASIS 5 8 4 1 3 6

56
11 OTHER 2 1 2 1 1

TOTAL 35 17 3 12 26 15 11 20

TABLE.5.CLINICAL PRESENTATION OF DIFFERENT

HEMATOLOGICAL DISEASES IN 51 CASES

SR.NO SIGN AND SYMPTOMS NO PERCENTAGES

OF OF CASES

CASES

1 Fever 35 68.62

2 Bone Pain 17 33.33

3 Stool OB 3 5.882

4 H/O Bleeding 12 23.52

5 Splenomegaly 26 50.98

6 Hepatomegaly 15 29.41

7 Jaundice 11 21.56

8 Lymphadenopathy 20 39.21

57
 80.00
68.63
70.00
60.00 50.98
50.00
39.22
40.00 33.33
29.41
30.00 23.53 21.57
20.00
10.00 5.88
0.00

In the present study the most common presenting complaint was of fever [35

cases(68.62%)], followed by splenomegaly [26 cases(50.98%)]and

lymphadenopathy [20 cases(39.21%)].

The hematological parameters observed in 51 cases with various hematological

disorders are shown in Table.6

TABLE.6: HEMATOLOGICAL PARAMETERS OF DIFFERENT HEMATOLOGICAL DISEASES IN 51 CASES

SR.N
O
DIAGNOSIS PARAMETERS
HEMOGLOBIN TC PLATELETS
4000
7
>12. 10- <400 - >1100 >1.
TO <7 <1.O
5 12.5 0 1100 0 0
10
0
1 MEGALOBLASTIC ANEMIA 2 8 3 4 3 3 7
APLASTIC/HYPOPLASTIC
2 3 4 6 1 7
ANEMIA
IDA/MICRONORMOBLASTI
3 C ERYTHROID 1 1 1
HYPERPLASIA
4 DIMORPHIC ANEMIA 3 1 2 3
5 ITP 1 1 1

58
 6 PLASMA CELL LEUKEMIA 1 1 2 1 1
7 MPD 1 1 1 1 2
8 LEUKEMIA 1 2 5 1 2 5 7 1
NORMOCELLULAR
9 4 1 2 1 4 2 7
MARROW
10 E/O METASTASIS 1 2 3 2 2 2 4 1 7
11 OTHER 1 1 1 1 1 1
TOTAL 2 8 17 24 15 18 18 21 30

TABLE.6 A:HEMOGLOBIN LEVELS IN 51 CASES

10- 7 TO
>12.5 <7
12.5 10
MEGALOBLASTIC ANEMIA 2 8
APLASTIC/HYPOPLASTIC
3 4
ANEMIA
IDA/MICRONORMOBLASTIC
1
ERYTHROID HYPERPLASIA
DIMORPHIC ANEMIA 3
ITP 1
PLASMA CELL LEUKEMIA 1 1
MPD 1 1
LEUKEMIA 1 2 5
NORMOCELLULAR MARROW 4 1 2
E/O METASTASIS 1 2 3 2
OTHER 1 1
TOTAL 2 8 17 24

59
 12
10
8
2
6 8 2
4 5
4 1 3

2 2 4 2
2 3 3 1 1 1
0 1 1 1 1 1 1 1

TABLE:6 B: TOTAL WBC COUNT (cells/cumm) LEVELS IN

51 CASES
4000
<4000 - >11000
11000
MEGALOBLASTIC ANEMIA 3 4 3
APLASTIC/HYPOPLASTIC
6 1
ANEMIA
IDA/MICRONORMOBLASTIC
1
ERYTHROID HYPERPLASIA
DIMORPHIC ANEMIA 1 2
ITP 1
PLASMA CELL LEUKEMIA 2
MPD 1 1
LEUKEMIA 1 2 5
NORMOCELLULAR
1 4 2
MARROW
E/O METASTASIS 2 2 4
OTHER 1 1
TOTAL 15 18 18

60
 12
10
8 3

6 1 2 4
4 5
4
6 4 2
2 2 2
3 2 1 2 1
0 1 1 1 1 1 1 1

TABLE :6C: PLATELET (lacs/cumm) COUNT IN

51 CASES
<1.O >1.0
MEGALOBLASTIC ANEMIA 3 7
APLASTIC/HYPOPLASTIC
7
ANEMIA
IDA/MICRONORMOBLASTIC
1
ERYTHROID HYPERPLASIA
DIMORPHIC ANEMIA 3
ITP 1
PLASMA CELL LEUKEMIA 1 1
MPD 2
LEUKEMIA 7 1
NORMOCELLULAR MARROW 7
E/O METASTASIS 1 7
OTHER 1 1
TOTAL 21 30

61
 12

10

8
1
6 7

4 7
7 7 7
2
3 3 1 1
2
0 1 1 1 1 1

Total 51 cases were diagnosed based on BMA,BMI and BMB are listed in Table 7.

TABLE.7: CASES DIAGNOSED ON BMA,BMI AND BMB

SR.NO DIAGNOSIS(TOTAL) BMA BMI BMB
MEGALOBLASTIC ANEMIA
1
(10) 8 9 9
APLASTIC/HYPOPLASTIC
2
ANEMIA (6) 4 4 6
IDA/MICRONORMOBLASTIC
3
ERYTHROID HYPERPLASIA (1) 1
4 DIMORPHIC ANEMIA (3) 3 2 2
5 ITP (1) 1 1 1
6 PLASMA CELL LEUKEMIA (2) 2 2 2
7 MPD (2) 1 1 2
8 LEUKEMIA (8) 8 4 4
NORMOCELLULAR MARROW
9
(4) 6 4 4
10 E/O METASTASIS (12) 4 4 6

62
 11 OTHER (2) 1 0 1
TOTAL 39 31 37
PERCENTAGE % 76.47 60.78 72.55

100%
90% 9 2 1 2 4 4
80% 6 2 6 1
70% 4
60% 2 4
50% 9 1 1 2 0
40% 4 1 4
30% 3 8 6 1
20% 8 4 1 2 1 4
10%
0%

BMA BMI BMB

TABLE.7A : CASES DIAGNOSED BY BMA

NO OF
SR.NO DIAGNOSIS CASES
1 MEGALOBLASTIC ANEMIA 8
2 APLASTIC/HYPOPLASTIC ANEMIA 4
IDA/MICRONORMOBLASTIC ERYTHROID
3
HYPERPLASIA 1
4 DIMORPHIC ANEMIA 3
5 ITP 1
6 PLASMA CELL LEUKEMIA 2
7 MPD 1
8 LEUKEMIA 8
9 NORMOCELLULAR MARROW 7
10 E/O METASTASIS 4
11 OTHER 1
TOTAL 40
PERCENTAGES (%) 79

63
 TABLE: 7B: CASES DIAGNOSED BY BMB
NO OF
SR.NO DIAGNOSIS CASES
1 MEGALOBLASTIC ANEMIA 2
2 APLASTIC/HYPOPLASTIC ANEMIA 3
3 MPD 1
4 E/O METASTASIS 4
5 OTHER 1
TOTAL 11
PERCENTAGE (%) 21

The histo-pathological findings and the diagnosis made on the biopsies were
compared with the findings on the BMA and BMI.

Further analysis of the data is done under discussion part.

Table .8 Various Disorders Diagnosed in 13 Cases of Pancytopenia in the Present

Study.

TABLE. 8: VARIOUS DISORDERS DIAGNOSED IN 13 CASES OF PANCYTOPENIA

NO OF PERCENTAGES OF
SR.NO DIAGNOSIS CASES OF
CASES
PANCYTOPENIA(%)
1 MEGALOBLASTIC ANEMIA 4 30.77
APLASTIC/HYPOPLASTIC 6
2
ANEMIA 46.15
3 DIMORPHIC ANEMIA 1 7.69
4 LEUKEMIA 1 7.69
5 E/O METASTASIS 1 7.69
TOTAL 13 100.00

64
Pancytopenia was more common in aplastic/hypo plastic anemia followed by

megaloblastic anemia.

Most common cause of pancytopenia in our study was aplastic/ hypoplastic

anaemia.

65
6. Discussion

The comparative evaluation of BMA, BMI and BMB was undertaken to assess

whether all three procedures are required in every case or in certain cases only

aspirate or only biopsy would give adequate diagnostic information, thereby

relieving the patient from unnecessary additional stress.

In thirty one cases all three procedures were performed. In rest of the cases

only one or two procedures were done.

The cases diagnosed on bone marrow examination were grouped into four
categories.

a) Cases where BMA was diagnostic and BMB didn’t revealed any
additional information: 39/51 (76.47%).

b) Cases where diagnosis was given on BMA alone, BMB was not
contributory due to inadequate or failed biopsy: 15/51(29.41%) .

c) Diagnosis on BMB alone where BMA was not contributory for diagnosis

due to dry tap or diluted marrow on aspiration: 11/51 (21.56%)

d) Cases where only aspirate was confirmatory : 40/51 (80%)

Thus BMA was diagnostic in 76.47% (39/51) cases. The BMI was adequate in

58.82% (30/51). Rest were found to be admixed with blood. BMI gave a better

indication of cellularity than BMA. The BMB was diagnostic in 21.56% (11/51)

cases in our study where BMA resulted in dry tap/diluted marrow on aspiration.

66
In the course of this study it was observed that it takes 6-7 procedures to learn

the proper techniques. The ‘’learning curve’’ includes proper sedation /co-

operation of patients, proper counselling of patient as well as their relatives too.

The observation by Cotelingam J.D.119 “Ideally the bone marrow core biopsy

should be reviewed with the knowledge of clinical history, complete blood

counts, peripheral blood picture and bone marrow aspirate smears” was

corroborated in this study.

In the present study fifty one patients diagnosed with various haematological

disorders were studied. The various diseases diagnosed by BMA,BMI and BMB

were megaloblastic anemia, aplastic/ hypoplastic anemia,

IDA/micronormoblastic erythroid hyperplasia, dimorphic anemia, ITP, plasma

cell leukemia, MPD, leukemia, normocellular marrow, e/o metastasis and others

listed in Table.9.

TABLE.9: THE DIAGNOSIS OF 51 CASES

SR.N DIAGNOSIS NO OF CASES PERCENTAGES

O OF CASES(%)

1 MEGALOBLASTIC ANEMIA 10 19.6%

2 APLASTIC/HYPOPLASTIC ANEMIA 7 13.7%

3 IDA/MICRONORMOBLASTIC ERYTHROID 1 2.0%

HYPERPLASIA

67
4 DIMORPHIC ANEMIA 3 5.9%

5 ITP 1 2.0%

6 PLASMA CELL LEUKEMIA 2 3.9%

7 MPD 2 3.9%

8 LEUKEMIA 8 15.7%

9 NORMOCELLULAR MARROW 7 13.7%

10 E/O METASTASIS 8 15.7%

11 OTHER 2 3.9%

TOTAL 51 100

Comparison of our study with the other studies is shown in the Table.9.

TABLE.9: Comparison of our study with the breakup of diagnosis of other studies

Dr.
Sabharwal by Dr. Pudasaini S.
Manjula
et al 50 Shweta 78 and Prasad PRESENT
SR.NO DIAGNOSIS et
(2009) (2011) K.B.R. 79 STUDY(N=51)
al.(2006)
(n=30) (N=50) (2012) (57) 80
.(n=50)

1 MA 1 (3%) 20 (40%) 19 (33%) 15 (30%) 10 (20%)

2 IDA 3 (6%) 4 (7%) 1 (2%)

3 DMA 8 (16%) 10 (20%) 3 (6%)

4 AA/HYPOPLASTIC A. 2 (7%) 3 (6%) 3 (5%) 2 (4%) 7 (14%)

5 SA 1 (2%)

68
6 ITP 1 (3%) 3 (6%) 6 (10%) 4(8%) 2(4%)

7 HYPERSPLENISM 3 (6%)

8 MM 3 (10%) 2 (4%) 2 (4%) 4 (8%) 2 (4%)

9 CML 4 (13%) 2 (4%) 1 (2%)

10 INFECTIVE 1 (2%) 8 (14%) 1 (2%)

PATHOLOGY/LEISHMANIA
11 IMF 7 (24%) 1 (2%) 1 (2%)

12 PV 1 (2%) 1 (2%)

13 NORMOCELLULAR 2 (7%) 2 (4%) 6 (10%) 3 (6%) 7 (14%)

14 ACUTE LEUKEMIA 5 (17%) 7 (12%) 4 (8%) 7 (14%)

15 MDS 3 (10%) 2 (4%) 1 (2%)

16 LYMPHOPROLIFERATIVE 1 (3%) 3 (6%) 5 (10%)

17 E/O METASTASIS 1 (3%) 4 (8%) 3 (6%)

30

25

20

15

10 SABHARWAL et al.(n=30)

5 SHWETA et al. (N=50)

PUDASAINI PRASAD(57)
0
MANJULA et al.(n=50)
PRESENT STUDY(N=51)

69
The variations in the percentages of diseases in different studies is due to less
number of sample size

Age wise distribution of different hematological studies are compared with

other studies in Table.10.

Table.10: omparision of age in different studies

SR. Age (years) Kibria G. et Dr. Shweta present study

NO al(2010) 81 78 et (n=51) (%)
(n=177) (%) al.(2011) (%)
1 <10 13 (7.34) - 13 (25.49)
2 11 20 40 (22.59) 12 (24) 9 (17.64)
3 21-30 34 (19.20) 14 (28) 7 (13.72)
4 31-40 23 (12.99) 11 (22) 7 (13.72)
5 41-50 16 (9.03) 6 (12) 1 (1.96)
6 >51 51 (28.82) 7 (14) 14 (27.45)
Total 177 50 51

35

30

25
Kibria G. et al(2010) 81
20 (n=177) (%)
Dr. Shweta 78 et al.(2011)
15 (%)
present study (n=51) (%)
10

0
<10 11 20 21-30 31-40 41-50 >51

In present study majority of the patients (29.41 %) were of >51 years of age.
Kibria G. et al(2010) 81 found the same (28.85%).

Comparison of sex distribution in different studies is shown in the table.12 below.

Table 12: Comparison of sex distribution in different

studies

70
Study M:F
Egesie et al(2009) 82 (n=105) 1.5:1
Gayathri et al(2011) 83 (n=104) 1.2:1
Kibria et al(2010) 81 (n=177) 1.0:0.99
Niazi et al(2004) 84 (n=89) 1.7:1
Jha et al(2008) 85 (n=102) 1.5:1
Pudasaini S. et al(2012)79 (n=57) 1:1.1
Dr. Shweta et al.(2011) 78 (n=50) 0.92:1
Present study 2:1

In present study male to female ratio was 2:1 which was near similar to studies
conducted by Niazi et al(2004) 84 where the male to female ratio was 1.7:1.

Table.12 : Comparison of diagnostic utility of aspiration and trephine biopsy

Study BMA (%) BMB (%)

Nanda A. et al(2002)14 88.6 11.4

Pandya A. et al(2012)15 70 30

Mahajan V. et al(2013)16 94 06

Smita Chandra et
70 30
al(2011)68

Present study 74 26

The study by Smita Chandra et al(2011)68 showed 78% positive correlation

between aspirate and biopsy and 84.3% between imprint and biopsy.

The present study observed that although the diagnostic yield of BMA was
highest (74.5%) but diagnostic yield of BMI was also considerably high (58.82%) in
comparison to BMA (74.5%) and BMB (70.58%) in diagnosing various
haematological disorders. This is in contrast to other studies which have
observed imprint cytology to be of limited valve except in cases of dry tap.50

71
Nanda A. and Basu S. et al(2002)14, in their study have found that aspiration
alone was sufficient in making a diagnosis in 88.6% cases, which as well
correlated with the diagnosis made on trephine sections. In the remaining 11.4%
cases, trephine biopsy was necessary for making a diagnosis due to incomplete
information provided by aspiration or its inability to give a correct diagnosis.
These cases were mostly hypoplastic, aplastic marrow, myelofibrosis and marrow
infiltration by metastatic tumors and lymphomatous infiltrations.

Pandya A. and Patel T. et al(2012)15, in their study found that aspiration alone
was sufficient in making a diagnosis in 70% cases. In these cases trephine biopsy
gave additional information. In the remaining 30% cases trephine biopsy was
necessary for making a diagnosis due to incomplete information provided by
aspiration or its inability to give a correct diagnosis.

In the present study of 51 cases, aspiration was diagnostic in 79% (40/51) cases
and trephine biopsy was diagnostic in 21% (11/51) cases where aspiration mainly
was a dry tap or diluted with blood.

It was found that complete clinical and other relevant parameters were needed
in evaluating the bone marrow aspiration smears and biopsy sections to arrive
at a conclusive diagnosis.

Sabharwal B.D. and Malhotra V. et al(1990)13 stated that core needle biopsy of
bone marrow is a valuable diagnostic aid for measurement of marrow
cellularity, metastatic tumors and fibrosis. It should not be taken as a substitute
for examination of the marrow by aspiration smear but is a complementary
procedure, which affords several advantages.

BMA was used to study morphology, maturation stages of blood cells,

differential counts, and assessment of myeloid to erythroid ratio. While trephine
biopsy gave information about cellularity, architecture and focal lesions like
granuloma.

PANCYTOPENIA

72
Table. :Various Disorders Diagnosed in 104 Cases of Pancytopenia in a
Study by B. N. Gayathri et al 71(2011)
Sr. Diagnosis No. of Percentage of
No. Cases Cases

1 Megaloblastic anaemia 77 77.04

2 Aplastic anaemia 19 18.26

3 Subleukaemic leukaemia 4 3.86

4 Malaria 2 1.93

5 Multiple myeloma 1 0.96

6 Storage disorder 1 0.96

TOTAL 104 100%

VARIOUS DISORDERS OF PANCYTOPENIA IN

2 1
THE STUDY BY B. N. GAYATHRI et al
1
4
19 Megaloblastic anaemia
Aplastic anaemia
Subleukaemic leukaemia
Malaria
77
Multiple myeloma
Storage disorder

73
The comparison of incidence of pancytopenia in different (Indian) studies is

shown in table-.

Table –Comparison of incidence of pancytopenia in different Indian studies

Sr. Study Total No. Of No. Of cases % of

No. of Pancytopenia
Cases Pancytopenia out of Total
Cases

1 Tilak et al 72(1999) 205 77 37.5%

2 Khodke et al 73(2001) 250 50 20%

3 Present Study 51 13 23.52%

In the present study 23.52% of patients had Pancytopenia which is comparable

to that of Tilak et al study72 (1999).

Further analysis of the data was done under the following headings:

Megaloblastic anemia
IDA/ micronormoblastic erythroid hyperplasia
Dimorphic anemia
Aplastic/ hypoplastic anemia
ITP
Multiple myeloma
CML
PV
Acute leukaemia
Polycythemia vera
Lymphoproliferative disorder
Normocellular marrow

74
Miscellaneous

MEGALOBLASTIC ANEMIA:

In the present study 19.6% (10/51) of cases had megaloblastic anemia. The male
to female ratio was 4:1 with male preponderance. The commonest presenting
complaint was fever 60 % (6/10) and splenomegaly 60 % (6/10). Pallor was
present in all the cases. Hepatomegaly was present in 30% (3/10) and
lymphadenopathy and h/o bleeding 20% (2/10) of cases.

It was most common in age group of 1-10 years. One case in each 11-20 years
and 21-30 years age group. 4 cases were in 31-60 years age group. BMA was
hyper cellular and showed megaloblastic hyperplasia and occasional giant
metamyelocytes. Trephine biopsy in all the patients showed erythroid
hyperplasia. In two cases, BMA shows evidence of intranuclear dog ear
parvovirus inclusions in erythroid precursors.

Laboratory Parameters

Study/Author % of Cases of Severe anaemia

Saina et al 73(2009) 69%

Present study 100%

Thus, in the present study severe anemia was present in all the cases while in
study by Saina et al73 (2009) it was present in 67% .

Age

Average age in cases of megaloblastic anaemia in different studies is shown in

the table:

Table. :Average age in cases of megaloblastic anaemia in different studies

Sr. No. Studies / Author Average Age

75
 1 Saina et al73 (2009) 38

2 Sarode et al 76(1989) 25

3 Ari Zirman et al 77(1983) 65

4 Rajashekar swamy et.al. 120 35

5 Suchitha S. et al 121 45

6 Manjula et al. 80 30

4 Present study 46

In the present study age group is comparable with the study of Suchitha S. et al.

Sex

The comparison of M:F ratio of cases of megabolastic anaemia in different

studies is shown in the table:

Table. : M:F ratio of cases of megabolastic anaemia in different studies

Sr. Studies / Author M:F
No.

1 Saina et al73 (2009) 1:1

2 Sarode et al 76 (1989) 1:1

3 Ari Zirman et al 77(1983) 1:1

4 Rajashekar swamy et.al. 120 1.3:1

5 Suchitha S. et al 121 1.3:1

6 Manjula et al. 80 2.3:1

4 Present study 4:1

76
In our study there is male preponderance. One of the reasons for it might be
males visit to hospital in our setup more compared to females, as people come
from poor socio economic families still believe in male dominancy.
Symptomatology

Table. :The comparison of sign and symptoms in cases of Megaloblastic

anaemia in different studies
Sr. Studies / Author Most common Most common
No. symptoms sign

1 Ari Zirman et 77 Weakness [39%] Pallor [54%]

al(1983)

2 Sarode et al 76 Fatigue [70%] Pallor [85%]

(1989)

3 Present study Weakness [all cases] Pallor [all cases]

Signs and symptoms in the present study are comparable with the study by
Sarode et al. 76

Association with Hepatomegaly And Splenomegaly:

The comparison of hepatomegaly and splenomegaly in cases of megaloblastic

anaemia in different studies is shown in the table:

Sr. Studies / Author Hepatomegaly Splenomegaly

No.

1 Ari Zirman et al 77 (1983) 34% 12%

2 Sarode et al 76 (1989) 34% 20%

3 Present study 30% 60%

In the present study splenomegaly was more compared to hepatomegaly.

Bone Marrow Findings:

77
BMA were available in 8 cases, BMI in 9 cases and BMB in 9 cases.

Aspirate smears: All the marrow aspirates showed hypercellular marrow particles
with decreased fat cells. There was pronounced megaloblastic erythroid
hyperplasia causing reversal of M: E ratio. Increased mitotic activity with
abnormal forms of mitotic figures was seen. Howell - Jolly bodies were also
present in some cases. Nuclear chromatin was open, particulated with
increase in the parachromatin spaces.

Myeloid series showed giant metamyelocytes and giant band cells.

Metamyelocytes with "C" shape or sigma shape nuclei were also present in
some cases.

These findings are comparable to that reported by Jacques mallarme74 (1948).

Ioannides K and Rywlin AM 44 stated that aspiration is useful in making out better
individual cell morphology whereas biopsy is useful in bone marrow architectural
pattern and distribution.

On comparing morphological features on BMI; cellularity was more appreciated

in BMI than in BMA. This nuclear abnormality i.e. particulate nuclear chromatin is
probably artifact of crushing. However, crushing had produced prominent
nuclear streaking, which was more pronounced near periphery of smears.

Trephine Biopsy Sections: 2 patients had dry tap. Trephine biopsy showed
hypercellular marrow with erythroid hyperplasia except in one case which was
diluted with blood. It was not possible to differentiate megaloblastic hyperplasia
from normoblastic [particularly macronormoblastic] hyperplasia on biopsies
except in very few cases with severe megaloblastic anemia. In such cases there
was increased erythroid cells which had large, round to oval nuclei with one or
more basophilic nucleoli near nuclear membrane.

78
Conclusions

Bone marrow aspirate is the best marrow preparation for the diagnosis of
megaloblastic anemia. But, the bone marrow biopsy was complementary to
aspiration for the diagnosis of megaloblastic anaemia in the 2 cases of dry tap
in our study.

IRON DEFICIENCY ANEMIA/ MICRONORMOBLASTIC ERYTHROID

HYPERPLASIA

Clinical profile

The prevalence of iron deficiency is high in people with low socio-economic

status. In the present study 2% (1/51) of patient had iron deficiency anemia.

She was 25 years female. The commonest presenting complaint was

breathlessness and fever. Pallor and splenomegaly was present.

Beveridge BR et al 122 found that out of 371 patients, 234(63%) patients

presented with symptoms of anaemia such as pallor, fatigue and lethargy,

59(61%) presented with symptoms of the disease causing anaemia and in the

remaining 78(21%) patients anaemia was discovered at the time of evaluation

for an unrelated complaint.

In the study by Mnjula et al80, 10(66.66%) patients presented with pallor,

7(46.66%) with fever, 4(26.66%) with fatigue and 6(33.33%) with

hepatosplenomegaly.

79
Laboratory Parameters

She had severe anemia. [Hb <7.0gm%] and peripheral smear suggested

microcytosis [MCV <76fl] and anisocytosis [RDW 29]. The case had low Serum

ferritin level. Hemoglobin electrophoresis diagnosis was sickle cell trait.

Considering the severity of Microcytosis , peripheral smear examination findings,

severity of anemia and low S. ferritin level iron deficiency anemia was

suspected.

Age and Sex

Table. :Average age of cases of iron deficiency anaemia in different studies

Sr. No. Studies / Author Average Age

1 Ayub et al86 (2005) 41-45years

2 Manjula et al 80 29.4

2 Present study 25years

In the present study age group is lower than in study by Ayub et al 86.

Table. :Comparison of M:F ratio in iron deficiency anaemia in different studies.

80
Sr. No. Studies / Author M:F

1 Ayub et al86 (2005) 1:1.5

2 Manjula et al . 80 2:1

2 Present study 0:1

In the present study M:F ratio is comparable with study by Ayub et al.86

Beveridge BR et al 122 in their study of 371 patients of hypochromic anaemia

found women to be affected in reproductive years, whereas incidence in men

increased with advancing age.

In the study by Manjula et al. showed male to female ratio was 2:1. Amongst

males one patient was in 11-20years, one was in 21-30years, two were in 31-

40years and the remaining above 40years of age, concluding that iron

deficiency increases with advancing age. Of the five females, 2(40%) were in

11-20years, 1(20%) in 21-30years, 1(20%) in 31-40years and 1(20%) in 41-50years of

age group. The average age being 29.4years. All were in reproductive age

group.

Hematological Parameters

81
Table. :Comparison of hematological Parameters in different studies

Sr. Author Hb MCV MCH MCHC Serum

No. ferrtin

1 Manohar et 6 76 20 27 8.94

al87(2000)

2 Moh. T. Javed et 9 90 25 28 -

al88(2000)

3 Present Study 3.6 53.2 12.2 22.9 6

Hematological parameters of our study are comparable to study by Manohar et

al (2000)86.

Bone marrow findings:

Aspirate smears:

Bone marrow aspirates: showed micronormoblastic erythroid hyperplasia.

Myeloid and megakaryocytic series were unremarkable. Marrow iron was

absent.

Trephine Biopsy Section: Due to inadequate sampling ,as the female patient

was too fatty, biopsy didn’t suggest micronormoblastic picture.

Study by Goddard et al89 (2006) tells that microcytosis is characteristic of IDA but

it also occur in thalassemia though RBC count is raised in thalalesmia.

82
Microcytosis may be absent in IDA if combined with folate deficiency and in

that case RDW is raised. Anaemia of chronic disease may also present with

microcytosis. Serum ferritin is most powerful test for IDA. A serum ferritin <12µg/dl

is diagnostic of iron deficiency anaemia and is raised in chronic disease.

Therapeutic response to 3 weeks of iron supplementation or bone marrow

aspiration are the only ways of conforming true deficiency.

Our study presented with microcytosis , raised RDW and S. ferritin <12ug/dl and

bone marrow picture supporting iron deficiency anaemia.

According to study by D. Mukhopadhyay et al 90(2002) Bone Marrow aspiration

and staining for iron provide a definite diagnosis of iron deficiency anaemia or

sideroblastic anaemia. It is considered standard for assessing iron status, but is

seldom necessary because of availability of serum ferritin assay and other non-

invasive procedures.

Lundin P 123 his study stated, aspiration and biopsy of marrow usually are

preferred for iron stores because they are probably safer and the technique is

more familiar to hematologists than that for liver biopsy.

Gale E et al 124 and Trubowitz S et al 125 found good correlation between

histologic grading and iron content in bone marrow. Krause JR et al75 found

majority of the patients showing mild positivity to nil iron stores on aspirated

smears as compared to biopsy sections which showed good positivity.

83
Conclusions:

Whenever clinical and laboratory data do not differentiate iron deficiency

anemia from anemia of chronic disorders, bone marrow examination should be

done. It should also be done in all the cases of pancytopenia to detect marrow

hypoplasia / aplasia. Aspirates should be done for detection of

micronormoblasts and sideroblasts and trephine biopsy is complementary

procedure.

DIMORPHIC ANAEMIA

[Iron deficiency & Megaloblastic anaemia]

Clinical Profile

In the present study 5.9% (3/51) cases were of dimorphic anaemia. Patients age

group range between 21 -60yrs and one <10years. Male : Female ratio was 1:2.

Most common presenting complaint was weakness and fever. Pallor was

present in all cases. Hepatomegaly was present in 1 case and splenomegaly

was present in 3 cases.

Laboratory Parameters

84
 Moderate anaemia was present in 7 cases. RDW was raised in all. Peripheral

smear showed dimorphic picture with microcytes , macrocytes and

anisopoikilocytosis.

Bone Marrow Findings

Aspirate Smears and Imprint smears: Showed micronormoblastic and

megaloblastic erythroid hyperplasia. Iron stores were decreased in all the cases.

Trephine Biopsy Sections: Showed erythroid hyperplasia with hypercellular

marrow.

Age:

Table. :Average age in dimorphic anaemia in different studies

Sr. No. Studies / Author Average Age

1 Beyan et al91 (2005) 44

2 Maria et al92 (2008) 22

3 Zuberi et al93 (2007) 33

4 Dr.Shweta et al 78 39

5 Present study 32

85
In the present study age incidence is comparable with study of Zuberi et al 93

(2007).

Sex

Table. :Comparison of male to female ratio in dimorphic anaemia in different

studies:

Sr. No. Studies / Author M:F

1 Maria et al92 (2008) 1.4:1

2 Zuberi et al93 (2007) 1.5:1

3 Dr.Shweta et al 78 5:3

4 Present study 1:2

In the present study male to female ratio is reversed with the studies by Maria et

al and Zuberi et al (2008).

Hematological Parameters

Table. :Comparison of hematological parameters in different studies

Sr. No. Author Hb MCV RDW

86
1 Beyan et al91 (2005) 9.1 67 18

2 Zuberi et al93 (2007) 11.7 85 20

3 Present Study 8.03 70.56 19.66

In the present study Hb was lower and MCV, RDW were correlated with other

two studies.

According to study by Beyan et al 91 (2005) simultaneous deficiencies of iron and

B12 are common .Morphological abnormalities in RBC vary greatly according to

severity of deficiency of iron and B12.Vitamin B12 deficiency mask the

morphologic changes of coexisting iron deficiency but RBC indices are in

favour of microcytosis and hypochromia.

In the present study indices are of iron deficiency with peripheral smear showing

dimorphic population and raised RDW consistent with clinical diagnosis.

In the study by Maria et al92 (2008) it was found that iron and B12 deficiency in

the population studied was due to malaria and nutritional deficiency. High

prevalence of malaria, anaemia, iron and folic deficiency indicate health and

nutrition problem of the community.

87
Conclusion :As malaria is also prevalent in our area and most of patients are

from low socioeconomic group, so it could be one of the major cause of

dimorphic anaemia. BMA is sufficient enough for diagnosis of dimorphic anemia.

APLASTIC/HYPOPLASTIC ANAEMIA

Clinical Profile

In the present study 13.7% (7/51) were cases of hypoplastic/apllastic anaemia.

Three of them were between the age group of 11 – 20 years and two were

between 21 – 30 years each. Two of them falls in >51 years of age group. M:F

ratio was 6:1.Most common presenting complaint was fever and weakness ,

three of them h/o bleeding and hepatomegaly each. Two had bone pain. And

one had bone pain. Most common clinical feature was pallor.

Laboratory Parameters

Patients had pancytopenia with RBC mass reduced, Peripheral smear showed

few macrocytes.

Table. :AGE & SEX wise comparision

Sr. no Author Average age M:F Ratio

{years}

88
1 Khodke et al95(2001) 26 1:1

2 Lewis et al94 (1965) 40 1:1

3 Daniel NM and Byrds L 126 65 1.4:1

4 Boon TH and Walton JN 127 65 1:1

6 Manjula et al. 80 32 1:1

3 Present Study 33.3 5:1

In the present study average age of our cases were comparable with the study

by Khodke et al95 and male cases were more than other two stydies.

Bone Marrow findings:

Aspirate, Imprint smear: Were scantily cellular with increase fat space.

Trephine Biopsy : Severely hypocellular marrow was observed. Scattered

erythroid islands were seen in-between bony trabeculaes . Biopsy is done to

assess the cellularity and rule out other causes like acute leukaemias, MDS,

lymphomas which can also be hypocellular on aspiration 55. The above findings

helped us to rule out other causes of hypocellular marrow and to diagnose

aplastic anaemia.

Discussion: In Lewis et al94 (1965) study 22 bone marrow out of 60 were either

normocellular or hypercellular. They found that in several cases cellularity of

second aspirate differed from the first. They stated that such variability might

89
occur with a small area of marrow. Criteria for the diagnosis of aplastic anaemia

in cases of hypercellular and normocellular marrow have not been mentioned.

Wintrobe (1999)96 stated that progressive hypocythemia alone should be

sufficient reason for the diagnosis of aplastic anaemia, provided leukaemia is

excluded.

In the present study peripheral pancytopenia,pausicellular bone marrow

aspirate and imprint with biopsy without remarkable pathology except severely

hypocellular and erthroid islands are considered as aplastic anemia.

Conclusion:

In our cases based on ratio of fat cells to total hemopoietic tissue present in

aspirate smear, it is possible to qualitatively divide marrow into hypocellular,

hypercellular and normocellular

BMB is the most important preparation for determination of marrow cellularity

and for diagnosis of aplastic anemia compared to BMI and BMA..

ITP

Clinical Profile

There was one case of ITP in the present study. She was 40 yeears female. She

presented with menorrhagia, polymenorrhea and dysmenorrhea.

Laboratory Parameters

90
Splenomegaly, hepatomegaly and lymphadenopathy were absent. Platelet

count was <1 lac/µl.

Peripheral smear: Showed severely decreased platelet and mild microcytosis

and hypochromia.

Bone Marrow Examinations:

Aspirate Smear: The smears were hypercellular with markedly increased number

of megakaryocytes and predominance of hypolobulated and hypogranular

forms. Myeloid and erythroid series were unremarkable.

Aspirate Smear : Increased megakaryocytes are observed very well.

Trephine Biopsy Section: Biopsy was hypercellular with increased

megakaryocyte.

Because repeated episodes of thrombocytopenia was preceded by high-grade

fever, diagnosis of acute ITP was obvious.

Discussion:

According to George et al 97 (1996), in the patients of isolated

thrombocytopenia with an otherwise normal hemogram, and without any

evidence of an underlying disorder by clinical evaluation, a presumptive

diagnosis of immune thrombocytopenic purpura can generally be made

without a bone marrow examination. Bone marrow examination should be

91
reserved for patients who do not respond to therapy, those over 60 years of age

and those in whom splenectomy is considered.

Halperin et al98 (1988) studied 127 children of presumed ITP and found that in

only five (3.9%) of them, bone marrow examination led to a discordant

diagnosis. All the five patients had clinical and / or laboratory features atypical

of acute ITP. They concluded that bone marrow aspiration should be reserved

for those patients with atypical features of ITP.

Conclusions: Bone marrow examination is not indicated for the differential

diagnosis of isolated thrombocytopenia particularly if there is no atypical

features on peripheral smear examination and if there is evidence of

spontaneous recovery. Bone marrow aspirate examination is as good a

diagnostic value as bone marrow biopsy, if not more, for the diagnosis of

immune thrombocytopenia.

PLASMA CELL LEUKEMIA

Clinical Profile

There were two cases of multiple myeloma in the present study. Male to female

ratio was 1:1. Both patients were in age group of >51 years. They had fever,

pallor & weakness. One patient had severe body ache. Both had bone pain as

92
well as hepatomegaly was also observed. There were no splenomegaly or any

lymphadenopathy.

Niti Singhal et al 101 reported 30(61.22%) males and 19(38.77%) females out of 49

patients. The median age was 57years(range 31-82years). The most common

symptom was bone pain in 22(45%) cases, pathological fractures in 17(34.7%)

and weakness and fatigue in 15(30.6%) cases. Skeletal roentgenographic

abnormalities were detected in 39(79.6%) cases. Bartl R et al 102 found most

common symptom as bone pain(56%), followed by weakness and fatigue(45%)

and weight loss(17%). Skeletal abnormalities were detected in 76% of the

patients. Of these 47% had lytic lesions and 20% had a combination of

osteoporosis, lysis and pathologic fractures.

Laboratory Parameters

In one case hemogram ,peripheral smear and skull x-ray were inconclusive. In

another case Plasmacytoid cells with clefted and folded nuclei were seen in

peripheral smear. ESR was markedly raised in both cases. Urine examination in

both showed increase in B.J.Protein and urine electrophoresis showed M - spike

in gamma region. X-ray skull showed lytic lesion.

Bone Marrow Findings:

93
Aspirate and Imprint Smear: Bone marrow aspirate were richly cellular. They

showed predominance of plasma cells >30% .Cells of erythroid, and myeloid

series were lesser in amount. However, they were cytologically normal.

Majority of plasma cells were large in size with low N/C ratio and diffuse nuclear

chromatin. However, prominent nucleoli were seen in few cells. The cytoplasm

of plasma cells were variable, basophilic to light eosinophilic. Cytoplasmic

eosinophilia corresponds to the amount of synthesis of carbohydrate containing

immunoglobulin by individual myeloma cells. Perinuclear hoff was universally

present in all the myeloma cells. Binucleated to multinucleated plasma cells and

plasmablasts were present. Mitotic figures were not seen.

Trephine Biopsy Section: Bone marrow biopsies were hypercellular, showing

predominantly interstitial infiltrate of myeloma cells with sheets of myeloma cells

both in the interstitial region and in the paratrabecular region. Residual areas of

hemopoiesis were present.

Bartl R et al54 in their study found bone marrow being involved in 613(91%)

cases. Niti Singhal et al87 in trephine biopsies found interstitial pattern of

infiltration in 19(39%) cases, nodular in 19(39%) and diffuse in 11(22%) patients. In

majority of cases(49%), the cell type was plasmacytic(24cases), plasmablastic in

10 and pleomorphic in 15 cases. Poorly differentiated cell type(plasmablastic)

had a diffuse or nodular pattern of infiltration, where as majority of well

differentiated cell type(plasmacytic) had an interstitial pattern.

94
Discussion

For the diagnosis of multiple myeloma two of the following three criteria must be

present99.

(1) More than 20% plasma cells in a bone marrow aspirate.

(2) A paraprotein in serum or urine and

(3) Osteolytic bone lesions or osteoporosis.

(4) The probability of myeloma is high if an IgG paraprotein exceeds

30gm/L or if an IgA paraprotein exceeds 10 gm/L.

These patients were investigated for all the three criteria and were found

positive.

Greipp100 (1985) defined criteria for myeloma cell typing and classified

myeloma based on enumeration of plasmablasts, immature cells, intermediate

cells and mature cells on bone marrow aspiration examination.

Criteria for Myeloma Cell Typing

(I) Mature Myeloma Cells

Dense chromatin clumping

Nucleus < 8 µm

Nucleolus < 1 µm

95
Cytoplasm well developed

Nucleus eccentrically placed with a prominent hoff

(II) Intermediate Myeloma Cells not fulfilling criteria for other types.

(III) Immature Myeloma Cells

Diffuse Chromatin Pattern

Nucleus > 10 µm or nucleolus > 2µm with

Abundant cytoplasm

Nucleus eccentrically placed with a hoff

(IV) Plasmablastic Cells

Same as immature cells but cytoplasm less abundant, nucleus centrally placed

with little or no hoff.

Majority of the plasma cells in our cases were of mature to intermediate type.

Immature myeloma cells were few in number. Russell bodies, Dutcher bodies,

cytoplasmic crystals were not seen.

Niti Singhal et al 101 in trephine biopsies found interstitial pattern of infiltration in

19(39%) cases, nodular in 19(39%) and diffuse in 11(22%) patients. In majority of

cases(49%), the cell type was plasmacytic(24cases), plasmablastic in 10 and

pleomorphic in 15 cases. Poorly differentiated cell type(plasmablastic) had a

96
diffuse or nodular pattern of infiltration, whereas majority of well differentiated

cell type(plasmacytic) had an interstitial pattern. In the present study, Trephine

biopsy showed diffuse and paratrabecular pattern of infiltration of plasma cells.

Bartl R et al102 in their study found bone marrow being involved in 613(91%)

cases.

Sailer M et al 103 and Bartl R et al 102 in their study have quoted that all these

histological parameters provide valuable prognostic information, where ever

other modalities like β2 microglobulin and IL-6 levels are not available.

Clinical findings, radiological and laboratory parameters clearly suggested

suspicious of plasma cell discrasias which was than confirmed by bone marrow

aspirate smear. Bone marrow biopsy gave supplementary information regarding

pattern of bone marrow involvement.

CHRONIC MYELOID LEUKEMIA

Clinical Profile

In the present study there was one case of 55 year old female with chronic

myeloid leukemia. Presenting complaints was fever. An important finding on

physical examination was pallor and hepato splenomegaly. Lymphadenopathy

was not present.

97
Laboratory Parameters

There was an accidental finding in CBC and peripheral blood smear as she was

admitted for gynecological problem. There was peripheral leukocytosis of

150000 cells / cu.m.m. and peripheral smear showed increase in number of

immature of immature myeloid series of cells predominant being myelocytes,

metamyelocytes, neutrophils and eosinophils. The percentages of blasts were

5%.

Bone Marrow Examination:

Aspirate smear: Bone marrow were aspirated with difficulty. Site selected was

anterior superior iliac spine(ASIS) as she was too obese with weight of 105 kg.

and we couldn’t find PSIS. They were hypercellular with pronounced myeloid

hyperplasia [i.e. markedly increased M: E ratio]. Erythroid precursors were

decreased. Megakaryocytes were unremarkable.

Clot process was hypercellular with loss of fat cells. There was marked myeloid

hyperplasia with left shift with megakaryocytic hypoplasia.

Trephine biopsy inadequate bony trabeculae

Imprint: blood picture

Discussion

Age

98
The average age group and M:F ratio in chronic myeloid leukemia in the

different studies .

Table. Age ad sex wise Distribution in Chronic Myeloid Leukiemia

Sr. No. Studies / Authors Average Age M: F ratio

1. Gralnick104 et al (1971) 42.6years 0.95:1

2 Rao et al 127 27-72 years -

3. Manjula et al 80 35 years 1: 0

4. Present Study 55 years 0:1

In the present study average age was higher.

Symptoms

In the Gralnick et al104 (1971) study majority of the cases had abdominal pain,

easy fatigability, weight loss and anorexia.

Our cases had symptoms of fever and easy fatigability.

Bone Marrow Findings: In a series of 143 patients of Chronic myeloid leukaemia,

Burkhardt105 et al (1982) found granulocyltic predominance [Chronic

granulocytic leukaemia] in 66 patients (46%) while mixed granulocytic

megakaryocytic variety [CMGM] was found in 77 patients (54%).

In the present study there was granulocytic predominance.

99
Knox106 et al (1984) compared their 28 cases of CML divided in CGL [15 case

and CMGM [13 cases].Although Knox106 et al (1984) classified their cases into

CGL and CMGM, they concluded that no single histological feature consistently

differentiate these two entity and differentiation of borderline cases are

subjective.

In our cases cytogenetic study for ph chromosome was not done. There was

predominantly myeloid hyperplasia and megakaryocytic component was

unremarkable. These finding suggest that our patient had CGL but not CMGM.

PC shepherd 107 et al (1987) found that CML can be diagnosed in great majority

of cases from morphological profile of presentation ,peripheral blood films, but

high quality Romanowsky staining is essential.

Conclusion: Bone marrow examination, both aspirate and biopsy, is needed to

detect blast crisis (aspirate) and myelofibrosis [Trephine Biopsy] in chronic

myeloid leukemia.

POLYCYTHEMIA VERA

Clinical profile:

In present study there was one case of polycythemia vera in 51 year old male.

Jaundice was present. Also shortness of breath was there. On examination

splenomegaly, conjunctival congestion and peripheral cyanosis was observed.

100
CXR findings showed bronchiectasis. h/o chronic smoking since 30 years was

present.

Laboratory parameters:

He has Hb of 19.4 gm % with hematocrit was increased to 59.9 %. TC was

increased and there was mild ‘shift to left’ with neutrophilia. Platelet count was

borderline increased. ESR was significantly diminished.

Age:

Table. :Average age of cases in PV in different study:

Sr no. studied Mean age

1 Dr Richard Draper et al. 108 60

2 Francesco Passamonti et al 109 52

3 Barillo et al110 74

4 Brain J. et al 111 60

5 Present study 51

In present study mean age is slightly below than other studies.

Sex

In our study also male predominance is observed compared with other studies.

Table . : sex wise distribution in different studies

Sr. no Study M:F ratio

101
1 Passamonti et al 109 1.12:1

2 Barillo et al110 2:1

3 Present study 1:0

Laboratory and clinical Parameters

Table. :

Sr.no Parameters (mean Francesco Shweta et al 78 Present study

value) Passamonti et

al 109

1 Hemoglobin(gm/dl) 19.3 19.7 19.4

2 PCV (%) - 58 59.9

3 RBC mass(Mil/ul) - 7.75 8.05

4 Platelets 2.93 5.2 4.02

(lacs/cumm)

5 WBC count(cells 7600 14600 14800

/cumm)

6 JAC 2(V617F) (%) 89 100 100

7 Splenomegaly (%) 23 100 100

Bone marrow findings

102
Bone marrow aspirate showed hypercellularity with trilineage hyperplasia,

predominantly composed of erythroid and megakaryocytic hyperplasia. Iron

stores were depleted.

Bone marrow imprint showed mainly hypercellularity . marrow cells were

admixed with blood, so obscured expected cells.

Bone marrow clot process showed mainly erythroid series hyperplasia and

megakaryocytic proliferation.

Bone marrow biopsy showed sinusoidal dilatation and hypercellularity. Erythroid

hyperplasia with normoblastic reaction with normal maturation. Myeloid

hyperplasia is also seen. Megakaryocytic hyperplasia with pleomorphism and

heterotopia in para-trabecular areas but not bizarre in loose clusters.

Discussion:

Diagnostic criteria 112

The proposed revised World Health Organization criteria for the diagnosis of PRV

requires two major criteria and one minor criterion, or the first major criterion and

two minor criteria.

Major criteria:

103
 o Haemoglobin of more than 18.5 g/dL in men, 16.5 g/dL in women, or

elevated red cell mass greater than 25% above mean normal predicted

value.

o Presence of JAK2 617V F mutation or other functionally similar mutations,

such as the exon 12 mutation of JAK2.

Minor criteria

o Bone marrow biopsy showing hypercellularity with prominent erythroid,

granulocytic, and megakaryocytic proliferation.

o Serum erythropoietin level below normal range.

o Endogenous erythroid colony formation in vitro.

Other confirmatory findings no longer required for diagnosis include:

o Oxygen saturation with arterial blood gas greater than 92%.

o Splenomegaly.

o Thrombocytosis (>400,000 platelets/mm3).

o Leukocytosis (>12,000/mm3).

o Leukocyte alkaline phosphatase (>100 units in the absence of fever or

infection).

In present study both of the major criteria were fulfilled with one minor criteria

also satisfied. EPO was not done though, the most probable diagnosis was PV.

Conclusion :

104
BMA was confirmatory in present study but BMB showed megakaryocytic

hyperplasia clearly and no fibrosis. So, BMA is superior for confirmatory diagnosis

than BMB. BMB is essential only if there is dry tap in the spent phase of PV(post

PVMF), where there is marked fibrosed marrow is a hall mark.

IDIOPATHIC MYELOFIBROSIS/ OSTEOMYELOFIBROSIS

Clinical Profile

In the present study there was a single case [2%] of myelofibrosis. She was

female of 34 years age and presented with weakness and epistaxis. On physical

examination he had pallor, hepatomegaly and splenomegaly

,lymphadenopathy ad bone pain.

Laboratory Parameters

1) Hemoglobin 6 gm% [severe anemia]

2) Total WBC Count 6000/µl

3) RBC Count 1.2 million / µl

4) PCV 20%

5) Platelet Count 28000 / µl

6) MCV 68 fl

105
Peripheral Smear Examination :

RBC: Anisopoikilocytosis, mild hypochromia, few normoblasts and teardrop cells .

Discussion

Age

The comparison of median age of idiopathic myelofibrosis in the different

studies .

Table. : Median Age In Idiopathic Myelofibrosis

Sr. No. Author Median Age (Years)

1 Hongyu113 et al (2007) 57

2 Abu Hilal M114 et al (2009) 60

3 Lohmann115 et al (1983) 60

4 Present Study 34

In the present study our case was younger as compared to other studies.

Sex

The comparison of M: F ratio of idiopathic myelofibrosis in the different studies .

Sex Distribution in Idiopathic Myelofibrosis

106
Sr. No. Author M:F

1. Abu Hilal M et al (2009) 114 2:1

2. Present study 0:1

Symptomatology

The comparison of symptomatology in idiopathic myelofibrosis in the different

studies

Table. : Symptomatology in Idiopathic Myelofibrosis

Sr. No. Author Symptoms

1. Abu Hilal M114 et Gastrointestinal bleeding. Abdominal

al (2009) pain,ascitis

2. Present study Weakness, fever, anorexia ,abdominal

pain and epistaxis

Splenomegaly

Splenomegaly was universally present in the studies of Lohman115 et al

(1983),Abu hilal114 M et al (2009) as well as in the present study.

Laboratory Parameters

107
The comparison of various laboratory parameters in idiopathic myelofibrosis in

the different studies.

Laboratory Parameter in Idiopathic Myelofibrosis

Sr. No. Authors Hb [gm%] WBC Platelet

Count Count

X103/µl X103/µl

1 Abu Hilal M 114 et 10 8800 2.25 lac

al (2009)

2 Lohman115 et al Hematocrit <30% 50000 <1lac

(1983)

3 Present study Hematocrit 6000 28000

30%.Hb 6gm%

Hemoglobin value of our case was the lowest.

WBC count of our case was comparable to Abu hilal 114 et al (2009) .

Platelet count of our case was comparable to that of Lohman 115 et al (1983)

study.

On peripheral smear examination anisopoikilocytosis and teardrop cells were

present in all the studies.

108
Bone Marrow Findings:

Aspirate Smear: Bone marrow aspirate was dry tap.

Bone marrow imprint were acellular smears

Trephine Biopsy Section: the hypercellualr marrow showed diminished erythroid

and myeloid precursors. Streaming of the cells was discerned. There was marked

fibroblastic prolifertation appreciated. Alternate areas of hemopoiesis and

fibrosis were also seen. Marrow sinusoids were markedly dilated with intraluminal

hematopoiesis. There was megakaryocytic hyperplasia with variation in cell sizes

with bizarre forms. They demonstrated abnormal lobulation, nacked nuclei with

hypercondensed chromatin. Also there were dwarf megakaryocytes present.

There was increased amount of trabeculaeted bone with an irregular pattern.

There were diffuse endosteal thickening of existing bony trabeculae,

appositional new bone formation and also formation of new bony trabeculae

(osteoid seams) .

Discussion: Pitcoch116 et al (1962) found diffuse fibrosis in all the 70 cases studied

by them. Our case also had fibrosis.

Buhr117 et al (2003), found atypical pleomorphic megakaryocytes with

hyperchromatic nuclei.

Our case also had similar findings.

109
According to Polycythemia Vera study group:

(1) The hemoglobin concentration and red cell mass not be elevated

(2) The Philadelphia chromosome be absent and

(3) One third of the bone marrow be occupied by fibrous tissue.

Our patient had Hb 6 gm%, PCV 20%, and diffuse marrow fibrosis. These

parameters correlate with those of Polycythemia Vera study group. However,

cytogenetic study for Philadelphia chromosome was not done, because

aspirate was dry tap. An Italian consensus118, conference criteria for

myelofibrosis are

Essential Criteria

(1) Diffuse fibrosis

(2) Absence of Ph chromosome Plus

Four minor criteria or Three minor criteria if splenomegaly is present

Minor Criteria

(1) Splenomegaly of any grade.

(2) Anisopoikilocytosis with teardrop cells.

(3) Circulating immature myeloid cells.

(4) Circulating erythroblasts.

110
(5) Presence of clusters of megakaryoblasts and anomalous

megakaryocytes in bone marrow sections.

(6) Myeloid metaplasia.

Our case fulfilled following criteria

Essential Criteria

(1) Diffuse marrow fibrosis

Minor Criteria

(1) Splenomegaly

(2) Anisopoikilocytosis and teardrop cells

(3) Circulating immature cells

(4) Circulating erythroblasts

(5) Pleomorphic megakaryocytes with hyperchromatic nuclei

However, our patient was not investigated for Philadelphia chromosome and

myeloid metaplasia.

Knox et al119 (1984) and Wintrobe stated that the most reliable test to

differentiate idiopathic myelofibrosis from chronic myeloid leukemia with fibrosis

is Philadelphia chromosome.

111
Bain et al99 (2000) stated that distinction between primary myelofibrosis and that

evolving out of other MPD is not possible on histological grounds alone and that

such distinction is not of clinical significance.

In a study by Sitalakshmi S. and Srikrishna A. et al(2005)62, 33 out of 176 patients

of myelofibrosis revealed dry tap on aspiration. 15% cases were categorised as

fibrotic phase of the disease in reticulin stained sections, while the remaining 85%

cases belonged to the cellular phase of myelofibrosis.

Conclusions: Peripheral smear examination and trephine biopsy is sufficient for

the diagnosis of idiopathic myelofibrosis (osteomyelofibrosis)

ACUTE LEUKAEMIA

Clinical profile

Among 7 cases of acute leukaemia, 02 ( %) were females and 05 (54%) were

male.

Table-17: Distribution of acute leukaemia according to age and sex

Acute Leukemias
Age (yrs) Male Female
AML ALL

<10yrs 02 01 00 03

11-20 03 01 01 03

112
Total 05 02 01 6

Table. :Comparison of incidence and sex distribution of AML and ALL.

Study AML(%) ALL(%) Male : Female

Sharma J. et al(2004)49
30.6 69.4 1.25:1
(n=36)

Present study (n=13) 14.28 85.72 2.5:1

Sharma J. and Mohindroo S.(2004)49 studied 36 cases of acute leukaemias and

recorded 20 (55.5%) cases of males and 16 (44.4%) females. The age ranged

from 1- 42 years. AML accounted for 11 (30.6%) cases and ALL for 25 (69.4%)

cases. In AML 4 (37%) patients were in 8-14 years of age group and 1(9%) was

42years of age. In ALL 11 (44%) patients were in 1-7years of age group and 2(8%)

patients in 36-42years of age group.

The findings were similar to that of the study conducted by Sharma J. and

Mahindroo S.(2004).49

AML

A patient of AML was 16 year female.

113
Bone marrow aspiration showed morphology of AML-M4

Special stain - SBB was done with positive result.

Trephine biopsy not done, as patient didn’t cooperated much.

ALL

Among six patients of ALL five were male and one was female. Three cases

were diagnosed in <10yrs and 11-20 yrs age group each.

Bone marrow aspiration and imprint showed increase in blast cells >25% in all

cases.

Trephine biopsy was hyper cellular and showed replacement of marrow cells by

blasts. In two Patients it was dry tap, and packed marrow was found.

Sitalakshmi S. and Srikrishna A. et al(2005)47 found that acute leukaemias were

diagnostic on aspiration alone, trephine biopsy provided additional useful

information which proved to be correct in our study. Wolf-Peeters C.D.E.(1991)48

in his study found that in acute leukaemia, the marrow cellularity and extent of

marrow replacement and quality of residual haematopoiesis are easier to assess

in sections than in smears.

Sitalakshmi et al35 found that acute leukaemias were diagnostic on aspiration

alone, trephine biopsy provided additional useful information. Wolf-Peeters CDE

36 in his study found that in acute leukaemias, the marrow cellularity, degree of

114
fibrosis, extent of marrow replacement and quality of residual haematopoiesis

are easier to assess in sections than in smears.

Navone R et al 65 concluded that the finding of a dry tap should never be

dismissed as being due to faulty technique and always needs a bone marrow

biopsy. In present study trephine biopsies were hyper cellular in all the four

patients and showed blast cells in patients who had dry taps on aspiration. Bone

marrow aspiration and trephine biopsy in cases of dry taps which correlated

with the above studies.

Conclusion:

BMA revealed definitive morphological type of blasts and so it is sufficient

enough for diagnosis of acute leukemia and BMB helped to assess cellularity

and identify blasts. That’s why it is necessary only when there is dry tap on BMA.

LYMPHOPROLIFERATIVE DISORDERS

In the present study there were 5/51 cases (9.80%) diagnosed as LPD.

Clinical profile:

There were two patients in age group of 31-40 and 41-60 years each and one
was in 21-30 years of age. M :F ratio was 1.5:1. Out of these cases all had bone
pain, three had splenomegaly, hepatomegaly and lymphadenopathy.

One patient was diagnosed as angioimmunoblastic T cell lymphoma and one

as follicular lymphoma based on lymph node biopsy histopathology section.

Trephine biopsy specimens are an integral part of the diagnosis, staging and
prognosis of patients with LPD.

Laboratory parameters:

115
Peripheral smear findings showed atypical lymphocytes.

Bone marrow examination:

Bone marrow aspirate revealed atypical lymphocytes and lymphoplasmacytoid

picture with eosinophilia in one case each and one with atypical hairy cells.
There was one case with dry tap.

Bone marrow imprint showed increased lymphocytes.

Bone marrow biopsy gave more important findings like cellularity, topography
and pattern of marrow involvement. There were three interstitial pattern, one
paratrabecular and one nodular pattern of marrow involvement was found.

In a study by Dee JW et al , 68 cases were Hodgkin’s disease and 166 were non-
Hodgkin’s lymphoma and age ranged from 2-87years. Trephine biopsy was
positive in 43 cases, 5 were inadequate and 23 cases were negative out of 71
cases. In NHL, lymphocytic lymphomas have largest initial incidence of marrow
involvement. Of 48 cases of lymphocytic lymphoma, diffuse pattern of infiltration
was seen in 10(21%) and nodular in 38(79%) patients.

Trephine biopsy was hypercellular with diffuse pattern of marrow infiltration by

lymphocytes. Bone marrow histologic pattern appears to be a better single
prognostic parameter than any one of the variables in current clinical staging
systems 82

Conclusion:

BMB is must for LPD.

116
METASTASIS

The study by Smita Chandra et al(2011)68 showed 78% positive correlation

between aspirate and biopsy and 84.3% between imprint and biopsy. 93.3%
cases of metastatic solid tumours were correctly diagnosed on imprint while only
70% cases were diagnosed on aspirate cytology.

NORMOCELLULAR MARROW.

In the present study there were two cases of normocellular marrow.

In one case bone marrow examination was done to find out Kala Azar infection
but bone marrow was normocellular and no parasite found.

In another case peripheral smear showed prominent macrocytes but bone

marrow was normocellular. Patient was alcoholic. Alcoholism explains
macrocytes in such patients

OTHERS

Kala azar INFECTIVE PATHOLOGY

In a study conducted by Pudasaini S. and Prasad K.B.R. et al(2012)23 infective

pathology was seen in 12.3% cases while leishmaniasis was seen in 1.8% cases.
Kibria G. and Islam M. et al(2010)22 found 2.82% cases of leishmaniasis. In present
study visceral leishmaniasis was found in 2% cases.

Megakaryocytic hyperplasia

117
7. Summary

118
8. Conclusion

Bone marrow biopsy is a safe and easy procedure with very less patient

discomfort. It is cost-effective and does not require sophisticated equipments. It

may be difficult to perform biopsy under local anaesthesia in unco-operative

cases. Bone marrow biopsy is the diagnostic investigation in dry tap cases like

Aplastic Anaemia, Myelofibrosis, MDS and metastatic tumors. It also helps in

monitoring patients with leukaemias. The cellular architecture is well preserved

when compared to bone marrow aspiration. Bone marrow aspiration shows

better cellular details when compared to bone marrow biopsy.

The advantages in correct diagnosis of a case by bone marrow biopsy in

conjunction with the clinical, haematological and aspiration study, far

outweighs the minor disadvantages with biopsy.

119
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British Journal of Hematology 2007, 139, 14–19.

2. Bender, G.A. (1966). Great Moments in History Trephining in Ancient, Peru. Parke,

Davis and Company, Northwood Institute Press, Detroit, 20–25.

3. Jackson, R. (1988) Doctors and Diseases in the Roman Empire. British Museum

Press, A Division of The British Museum Company Ltd, 46,Bloomsbury Street,

London, pp. 117–118.

4. Jackson, R. (1988). Doctors and Diseases in the Roman Empire. British Museum

Press, A Division of The British Museum Company Ltd, 46,Bloomsbury Street,

London, pp. 117–118.

5. Dr.(lt col.) R.N.Verma - Manual And Color Atlas of Bone Marrow Biopsy. Biopsy

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135
10. Annexures

136
11. Proforma

137
12. Consent form

138
13. Master chart

139