Bone Marrow Interpretation

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Interpretation of Bone marrow aspiration and biopsy

Bone marrow is the site where blood cells develop from totipotential haematopoietic stem cells. It consists of red marrow (haematopoetic tissue ) & yellow marrow (fat ) in a meshwork of vascular sinuses and highly branched fibroblasts .

Production of blood cells commences in the yolk sac of embryo ,followed by liver & to some extent spleen from 2 months of gestation till birth and bone marrow after 7 months which forms the only important site after birth .

Bone marrow is found in practically all bones at birth ,but in adults it is confined to the central skeleton ( vertebrae, sternum, ribs, skull, sacrum, pelvis ) and proximal ends of long bones (femur, tibia ,humerus).

Bone marrow aspiration is the removal by suction of the marrow .  It is a simple and safe procedure.

 Individual cell morphology is well preserved

 But arrangement of cells in marrow & relationship between one cell & another is disrupted.

So this is an important tool for cell morphology but not for architecture

Bone marrow biopsy or needle core biopsy, is the removal of a small piece of intact bone marrow

 Considered better than aspiration for evaluation of cell distribution and relationship between different cell types.

 An important procedure when disease process is focal in nature

Bone marrow evaluation is not a baseline test, but rather a confirmatory test used to rule in or rule out specific hypotheses based on routine hematological investigations.

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Indications of bone marrow examination  Investigation of unexplained cytopenias

o Unexplained leukopenia (neutropenia). o Unexplained thrombocytopenia. o Bicytopenia or pancytopenia.  Anemias esp. Megaloblastic anaemia

 Investigation of atypical cells in the peripheral blood o Immature hematopoietic cells (blasts) in the blood

o Atypical RBC (e.g., basophilic stippling, multiple Howell-Jolly bodies), WBC, or platelet morphology.

 Persistent, unexplained marked increases in RBC, WBC, or platelet numbers in the peripheral blood.

 Evaluation of infectious diseases such as leishmaniasis or histoplasmosis.  Investigation of hematological neoplasia

o Differentiation, diagnosis, and staging of leukemias and lymphomas. o Diagnosis and staging of other neoplasias e.g. metastatic carcinoma.  Fever of unknown origin.

 Immunophenotyping 

Note :Bone marrow examination is not mandatory for diagnosis of leukaemias but is performed for subtyping , baseline blast cell percentage and cytogenetic analysis.

Same is true for diagnosis of iron deficiency anaemia where simple biochemical tests along with peripheral blood findings are sufficient for diagnosis.

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Specific indications of bone marrow biopsy  Dry tap

 Suspected aplastic anaemia  Metastatic deposits

 Myelofibrosis  Hairy cell leukaemia

 Inflammatory conditions like Granulomatous inflammation  Lymphoma infiltration ( Hodgkins & Non hodgkins lymphoma)  To see pattern of involvement in Chronic lymphocytic leukaemia

Preferred sites for bone marrow aspiration & biopsy

 Posterior superior iliac spine (both aspiration and biopsy)  Sternum (aspiration only in adults)

 Anterior iliac spine (both aspiration and biopsy)  Tibial tuberosity (aspiration in children <one year age)

Procedure ( from posterior superior iliac spine [PSIS] ) 1. Position patient in lateral decubitus

position

2. Prepare and sterilize the site with iodine solution

3. Anesthetize skin and bone with 2% lidocaine

4. Insert bone marrow aspiration needle 5. Syringe for aspirating bone marrow while aspiration needle is in place with trochar withdrawn

6. Aspirate is put on slide

7. Aspirate smear is made using another slide.

8. Biopsy is performed using the same skin

puncture but a different site (on same PSIS) than aspiration . 9. Feel the give of the needle as it enters the cortex

10. Withdraw the trochar from the needle

11. After another centimeter push, rotate the needle to cut the end of the specimen

12. Withdraw the needle with the specimen inside the needle

13. Push the biopsy specimen from the marrow end to the hub end with the trochar

14. Place the biopsy specimen in fixative (such as zenker’s) for decalcification and processing .

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Bone marrow aspiration & biopsy needles

Kilma aspiration needle

Salah aspiration needle

Jamshidi biopsy needle

Bone marrow aspiration film

 Stained with Wright stain or Wright-Giemsa stain, Prussian blue stains.  In addition, special stains may be used that aid in the classification of

malignant white blood cells.

 Satisfactory films: when marrow particles and free marrow cells can be seen  Differential counts should be made in the cellular trails

The bone marrow biopsy material is sectioned onto glass slides and stained with hematoxylin-eosin stain .

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Bone marrow evaluation includes the examination of : 1) Bone marrow aspirate;

2) Bone marrow core biopsy 3) Peripheral blood.

When a high-quality bone marrow aspirate is obtained, and depending on the reasons for doing the bone marrow examination, sometimes an aspirate (without a core) is sufficient for evaluation.

Peripheral blood (i.e., a CBC) ALWAYS is needed for accurate interpretation of bone marrow

Bone marrow interpretation :

The protocol

 Review patient history and laboratory data .

 Examine peripheral blood smear

 Examine bone marrow aspirate -Examine at least two films

-Low magnification (10X) examination - cellularity, megakaryocytes , infiltrates

-High magnification (100X) examination- Examine each cell line ,fine cytological details, differential count in cell trails

 Examine bone marrow biopsy

-Examine slides at minimum three levels

-Low power examination: evaluate cellularity , megakaryocytes , focal /diffuse nature of the lesion

-High power examination : evaluate each cell line , detail of focal lesion

 Evaluate flow cytometry , immunohistochemical stains etc.

 Assign final diagnosis.

A) Patient information

 Patient identification a. Name b. Identification number c. Age d. Gender

 Referring physician

 Date of procedure

 Clinical information

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 Constitutional symptoms- fever, fatigue,night sweats,weight loss

 H/o autoimmune disorder,

 Immune status i.e HIV

 H/o bleeding from any site

 Current medication –e.g aspirin

 Prior diagnosis-documented malignancy

 Prior therapeutic regimens

 Any transplantation

 Prior surgery- splenectomy,gasterectomy

 Physicalfindings-pallor,icterus,ecchymosis,any lymphadenopathy,hepatomegaly or splenomegaly

 Relevant laboratory data o ESR

o Biochemical investigations

o radiographic data & imaging studies

B) Peripheral blood film examination

Peripheral blood sample should be obtained on the same day as marrow collection. This is essential because rapid changes can occur in peripheral blood counts and accurate interpretation of cells in the marrow require knowledge of CBC results.

 Hemoglobin

 TLC

 DLC

 Platelet count

Microscopic description of RBCs ,WBCs & Platelets:

 number

 size

 shape

Peripheral smear interpretation

C) Reporting of a bone marrow aspirate

1) Specification of site : Posterior Superior Iliac Spine or Sternum.

2) Overall sample quality/Adequacy of specimen i.e whether fragments present or not seen as glistening particles caused by fat in the particles

3) Low power examination : Choose several of the best spread fragments which contain easily visible marrow particles

i) Particle cellularity (hypocellular, normocellular, hypercellular).Cellularity is assessed in relation to the amount of adipose tissue in the particles and can be estimated as a percentage . A rough estimate is made as follows :

Amount of haematopoietic tissue / amount of adipose tissue X 100.

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Cellularity averages 79%( haematopoietic tissue) at ages 0-9 yrs. 50% (haematopoietic tissue)at ages 30-69yrs. 29% (haematopoietic tissue) at ages 70-79yrs Normocellular marrow Hypocellular marrow : causes  Aplastic anaemia

 Inherited bone marrow failure syndromes

 Chemicals like Benzene

 Drugs like carbamazapine

 viruses,

 ionizing radiation

 Autoimmune disorders

 Paroxysmal nocturnal hemoglobinuria

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Hypercellular marrow Causes  myeloproliferative disorders,  myelodysplasia or other ineffective hematopoiesis,  leukemias,  lymphoma infiltration

 megaloblastic anemia, sidero blastic anemia,

 compensatory hyperplasia after cell destruction

Scanning under low power reveals a heterogenous population of cells composed of erythroid cells, cells of myeloid series, lymphoid cells, plasma cells etc.

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ii) Megakaryocytes. These are the largest cells randomly distributed throughout the bone marrow . These are assessed for :

a) Number (adequate/ inadequate): These make about 0.5% of total cells in the marrow . Roughly at least five megakaryocytes should be present per fragment b) Maturation (mature forms should exceed immature forms in normal marrow):

normal maturation of megakaryocyte includes following stages

o Megakaryoblast : 6-24 micrometer in diameter with large minimally indented nucleus having fine chromatin , multiple nucleoli and scant basophilic cytoplasm. Comprise 20% of megakaryocyte lineage cells in marrow.

o Promagakaryocyte : larger than megakaryoblast lobulated nucleus ,with comparatively coarser chromatin and more abundant and less basophilic cytoplasm than megakaryoblast .Comprise 25% of marrow megakaryocytes.

o Mature magakaryocytes measures 50-150 microns in size with abundant pink cytoplasm containing numerous purple-red or pink granules; nucleus has 8, 16 or 32 overlapping lobes ; no nucleolus. Megakaryocytes producing platelets may have demarcated granular clumps of platelets streaming from the margin

c) Morphology: normal morphology is described above

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Abnormalities in number Megakaryocytic hyperplasia

i) Reactive,in chronic inflammatory disorders ii) Idiopathic thrombocytopenic purpura (ITP) iii) Myeloproliferative diorders

iv) Essential thrombocythemia Megakaryocyte depression

i) Aplastic anaemia ii) Drugs

iii) Viral induced Abnormalities in maturation

Presence of young forms in Idiopathic thrombocytopenic purpura Abnormalities in morphology

i. Dysplastic megakaryocytes seen as hypolobulated micromegakaryocyte or nonlobulated nuclei in megakaryocytes of all sizes and multiple ,widely separated nuclei- In myelodysplastic syndromes(MDS)

ii. Hyperlobated nuclei – in Essential Thrombocythemia.

iii. Atypical megakaryocytes –enlarged with abnormal chromatin clumping associated with balloon shaped lobulation of nuclei as well as frequent naked megakaryocytic nuclei seen in Chronic idiopathic myelofibrosis .

iii) Search for abnormal cells i.e. Clumps of cells foreign to the bone marrow  Granulomas

 Metastatic deposits

 Macrophages

 Sea blue histiocytes in storage disorders

 Necrosis: leukemias and lymphomas, chemotherapy, AIDS

 Gelatinous transformation :anorexia, alcohol, AIDS

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4) High power examination : The part of the smear spread best is examined at high magnification

 A minimum of 500 nucleated cells should be evaluated under oil immersion.

 Differential using categories : erythroid, myeloid ,lymphoid and plasma cells is generally adequate

 Only intact cells are evaluated; bare nuclei should be excluded

 Counting is performed where cells are not overlapping, clustered or artifactually distorted

i) The myeloid:erythroid (M:E) ratio

Myeloid series : Neutrophil ,Eosinophil, Basophil precursors and mature forms are collectively reffered to as myeloid cells

a. Erythroid hyperplasia(M:E,1:1 or less)

-DNA synthesis defects(B12 ,folate deficiency, drugs like methotrexate)

-Hemoglobin synthesis disorders(Thalassemias & congenital sideroblastic anemia)

-Hemolytic anemias i.e hemogobinopathies,RBC membrane disorder or enzyme deficiencies) or acquired (immune hemolytic anemia,PNH)

-After massive haemorrhage -Polycythemia

Erythroid hyperplasia

b. Erythroid hypoplasia : Absence/suppression of red cell precursor  Pure red cell aplasia

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Congenital : Diamond blackfann syndrome

Acquired:Parvovirus B19 infection , Secondary to Thymoma etc. c. Myeloid hyperplasia  Myeloproliferative disorders  Myelodysplastic syndromes.  Acute leukemia  Infections Myeloid hyperplasia d. Myeloid hypoplasia

 Drug induced- cytotoxic drugs

 Chemicals like benzene etc.

 Virus induced

ii) Erythroid cell line. Evaluated for numbers, maturation, morphology. Erythroid cells in bone marrow are the erythroblasts in different stages of maturation seen as proerythroblasts, basophilic erythroblasts , polychromatic erythroblasts and orthochromatic erythroblasts.

Number : The mean percentage of erythroid series in bone marrow is about 25%

Cell composition of erythroid series with normal range

 Proerythroblasts 0.1-1.1%

 Basophilic erythroblasts 0.4-2.4%

 Polychromatic erythroblasts 2-30%

 Orthochromatic erythroblasts 2-10%

Erythroid hyperplasia and hypoplasia can occur in a variety of conditions as discussed in previous section on M:E ratio.

Maturation and morphology

Red cells are produced by proliferation and differentiation of erythroblasts in the marrow . During the course of differentiation, the size of erythroblasts progressively decreases and the character of nucleus and cytoplasm changes as depicted in the diagram below

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1.Proerythroblast 2.Basophilic erythroblast 3.Polychromatic erythroblast

4.Orthochromatic erythroblast 5.Reticulocytes 6.Red blood cells

Proerythroblast : These are the least mature of the morphologically identifiable cells of erythroid series measuring 14-20 micrometer in diameter , have round to oval nucleus with fine chromatin ,multiple nucleoli, and basophilic cytoplasm.

Basophilic erythroblast : It is a round cell measuring 12-16 micrometer in diameter (smaller than han proerythro blast) with coarser and more basophilic nuclear chromatin and more basophilic cytoplasm than proerythroblast.

Polychromatic erythroblast : It is a round cell 12-14 micrometer in diameter having coarse nuclear chromatin seen in deeply basophilic clumps and polychromatic cytoplasm because of acidophilic haemoglobin (starts appearing at this stage) and basophilic ribonucleic acid. The prolifertive activity of nucleus ceases after this stage.

Orthochromatic erythroblast : These measure 8-12 micrometer in diameter with a small pyknotic nucleus giving a homogenous blue-black appearance and predominantly acidophilic cytoplasm.

Reticulocytes are formed from Orthochromatic erythroblasts after extrusion of nucleus from latter .These than enter the blood and mature into red blood cells .

Abnormalities in maturation and morphology

1)

Megaloblastic maturation : Seen in megaloblastic anaemia .The changes occur at all stages of maturation and are best appreciated in late precursors . 1 4 2 5 2 6 2 2 3 2

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The cells are larger than the normal counterparts with an increase in both cytoplasm and nuclear size .The nuclear chromatin is arranged in fine reticular fashion to give a stippled appearance (sieve like).This stippled appearance is still well marked in polychromatic erythroblasts and sometimes also in orthochromatic erythroblasts

-Nuclear cytoplasmic asynchrony : the maturation of nucleus lags behind

cytoplasmic maturation

2) Dyserythropoiesis : seen in myelodysplastic syndromes ( primary and therapy related MDS )-seen as Nuclear alterations including budding, internuclear bridging ,karyorrhexis, binucleation , multinucleation and megaloblastoid change ( chromatin pattern as megaloblast but normal in size)

Cytoplasmic features including ring sideroblasts ,vacuolization and periodic acid schiffpositivity-

Bone marrow smear exhibiting 1) nuclear budding and 2) multinucleation in erythroid series of cells

3) Micronormoblastic maturation: Smaller than normal with scant cytoplasm

having ragged borders .Cytoplasmic maturation lags behind nuclear maturation so that nucleus often appears pyknotic despite the fact that cytoplasm is still polychromatic ( see normal maturation and morphology of erythroblasts) -seen in Iron deficiency anaemia

4) Maturation arrest : Characterized by an increase in the proportion of more primitive cells – seen in Megaloblastic anaemia , where promegaloblasts and basophil megaloblasts may constitute > 50% of erythroblasts .

iii. Myeloid cell line

. Neutrophil ,Eosinophil, Basophil precursors and mature forms are collectively reffered to as myeloid cells . These are evaluated for numbers, maturation and morphology.

Number : The mean percentage of myeloid series in marrow is about 55% . Neutrophil series constitute approximately 90-95 % of the total myeloid series and eosinophils make virtually all of the remainder.

Neutrophil series %

1

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– Myelocytes 5-23

– Metamyelocytes 7-27

– Polymorphs 4-28

 Eosinophil series 0.3-4

 Basophil series 0-0.5

Myeloid hyperplasia and hypoplasia can occur in a variety of conditions as discussed in previous section on M:E ratio.

Maturation and morphology

: Mature granulocytes are produced by the proliferation and maturation of precursors from the earliest recognizable stage , the myeloblast, through the promyelocyte, myelocyte, metamyelocyte and stab form stage ,until the mature segmented stage is reached. .Development of the three cell lines of myeloid series follows a similar pattern except that the distinction between colour of granules becomes obvious at the myelocyte stage.

The myeloblast

: measures 15-20 micrometer , with round to oval nucleus having fine nuclear chromatin , prominent nucleoli and moderate amount of basophilic cytoplasm .No granules are seen .

The Promyelocyte :features are similar to those of myeloblast except for the development of some cytoplasmic granules , coarser chromatin and prominent nucleoli.

The myelocyte

: It is slightly smaller than promyelocyte having prominent cytoplasmic granules( may be neutrophilic, eosinophilic or basophilic) and less basophilic cytoplasm with comparatively low nuclear cytoplasmic ratio , coarser chromatin than promyelocyte and no nucleoli. This stage is the most mature proliferating cell in the myeloid lineage . Subsequently steps in the maturation process occur in non-proliferating cells

The metamyelocyte

: features are similar to those of myelocyte except that nucleus is indented (kidney shaped) ,granules more prominent, cytoplasm less basophilic and chromatin more coarse.

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The band or stab form stage .

:characterized by indented nucleus with degree of indentation more than 50% of the nucleus diameter and cytoplasm colour identical to that of a mature segmented neutrophil .

Segmented granulocyte :

measures 12-14 micrometer in diameter having lobulated nucleus with lobes of clumped chromatin linked by a thin chromatin strand .

Mature neutrophil

has 2-4 lobes of nucleus .It measures 12-14 micrometer in diameter and pink cytoplasm with fine granules

Mature eosinophil

has bilobed nucleus ,measure 12-17micrometer in diameter with cytoplasm packed with distinctive spherical eosinophilc granules

Mature basophil

has nuclear lobes that tend to fold on each other resembling a closed lotus flower .It measures about 16 micrometer in diameter with distinctive large ,variably sized dark blue or purple granules in the cytoplasm obscuring the nucleus .

1. The myeloblast 2. The Promyelocyte 3. The myelocyte

4. The metamyelocyte 5. The band or stab form 6. Segmented granulocyte

Abnormalities in maturation and morphology

1

2

4

3

5

6 Basophil series

Neutrophil series

Eosinophil series

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 Increase in myeloid blasts : in acute myeloid leukaemias

The blast cells are lage with nucleus having fine chromatin prominent nucleoli and moderate cytoplasm with som e cells exhibiting granules and auer rods

.

 Morphology

Giant metamyelocytes and stab forms seen as large cells with nucleus having irregular outline in megaloblastic anemia

Dysplasia in myeloid series : seen as hypogranulation, nuclear hyper and hyposegmentation , marked left shift in maturation-MDS

A hypogranular, hyposegmented

neutrophil(pseudo-Pelger-Huët anomaly).

Note the severe thrombocytopenia.

A giant myeloid precursor.

Erythroid and myeloid precursors.

Many of the myeloid cells are

hypogranulated.

Some of the late

erythroid precursors are mildly enlarged.

Ring sideroblasts and increased

iron incorporation in both erythroid

A hypogranular, hyposegmented

neutrophil(pseudo-Pelger-Huët anomaly).

Note the severe thrombocytopenia.

A giant myeloid precursor.

Erythroid and myeloid precursors.

Many of the myeloid cells are

hypogranulated.

Some of the late

erythroid precursors are mildly enlarged.

Ring sideroblasts and increased

iron incorporation in both erythroid

Bone marrow aspirate showing hypogranulated neutrophils with left shift seen as increased number of myelocyte and metamyelocytes

iv. Maturation index.

 The proportions of proliferating ( proerythroblast+ Basophil+ polychromatic

erythroblast for Erythroid series and myeloblast + promyelocyte+ myelocyte in myeloid series ) vs nonproliferating cells ( orthochromatic erythroblasts in erythroid series and metamyelocyte+band form+ mature forms in myeloid series) can be estimated or counted for calculation of an erythroid maturation

index, myeloid maturation index, and overall ( adding the two) maturation index.

 Maturation indices are useful for semi-quantifying left or right shifts in hematopoietic cell maturation.

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v

Lymphocytes:

Normal range : 5-24% of total cells in marrow . In children may constitute up to 50% of all nucleated cells. Lymphocytes pass through a series of changes in the course of development from lymphoblasts.

Lymphoblasts

are slightly smaller than the myeloblasts except that the ratio of the diameter of nucleus to that of the cell tends to be greater and the number of nucleoli per nucleus are fewer than in the myeloblast .

Small lymphocyte:

These are small cells with a thin rim of cytoplasm , occasionally containing fine azurophilic granules. Uniform nuclei 9-12 micrometer in diameter which is roughly the size of a red blood cell.

Large lymphocyte

measuring 12-16 micrometer in diameter with round nucleus and clumped chromatin with more abundant pale blue cytoplasm containing azurophilic granules.

Evaluated for number and maturation.

Number :

Increased lymphocytes can be seen in

 Older individuals

 Chronic inflammatory & autoimmune diseases  Lymphproliferative disorders involving marrow

Maturation

 Presence of lymphoblasts : seen in leukaemias .

VI Plasma cells

 The plasma cells are seen as round to oval cells with 2 -3 times the size of small lymphocytes ,one or more eccentrically placed nuclei ,clumped chromatin in the form of cart wheel and a perinuclear clear zone

 These make 0-3.5% of total cells in the marrow.

 Evaluated for number , immature forms , inclusion .

Lymphoblasts : seen as cells having altered nucleocytoplasmic ratio , with fine nuclear chromatin ,inconspicuous nucleoli and scant amount of cytoplasm

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Neoplastic –Multiple myeloma .  Immature forms (plasma blasts) seen as pleomorphic

frequently multinucleate cells having dispersed nuclear chromatin, prominent nucleoli and high nucleocytoplasmic ratio. Seen in Multiple myeloma.

 Inclusions :seen because of condensed or crystallized cytoplasmic Ig producing a variety of morphologically distinctive findings e.g

- Cherry red refractive round bodies -Russell bodies, - Vermillion staining glycogen rich Ig - Flame Cells,

- Multiple pale bluish white ,grape like accumulation-Mott Cells, - Overstuffed fibrils –Gaucher like cells, thesaurocytes.

Plasmablasts in multiple myeloma Mott cells

vii. Monocytes and macrophages

 These are the phagocytic cells involved in host defence against infection.

 These develop from monoblasts through promonocytes.

- Monoblasts are the least mature of the morphologically recognizable members of the monocyte-macrophage series. These are similar in appearance to myeloblasts

- Promonocytes: similar in size to the promyelocyte ,but has a more irregularly shaped and often deeply cleft nucleus containing nucleoli with cytoplasm containing granules which are more basophilic than in mature monocyte.

Monocytes:

Normal range : 0-0.6% of total cells in marrow



These are similar to the circulating leucocytes measuring 15 -18 micrometer in diameter and have large curved often horseshoe shaped (wrung towel) with bluish cytoplasm containing fine reddish granules

Increased in

 Reactive monocytosis  Acute monocytic leukaemia

 Chronic myelomonocytic leukaemia

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 These range from 15-80 micrometer having one or more oval nuclei and abundant cytoplasm with an irregular cytoplasmic outline containing granules and sometimes vacuoles which may contain phagocytosed material.

Increased production

o when there is an increased need for phagocytes.  Infectious process  During chemotherapy o Haemophagocytic syndromes o Storage disorders o Malignant histiocytosis

Cytochemical stains:

Cytochemistry is the technique used to identify diagnostically useful enzymes or other substances in the cytoplasm of the haematopoietic cells .It is particularly useful to characterize the blasts cells in acute leukaemias as myeloid ,to identify monocytic and granulocytic components in AML .

Various cytochemical stains used and the cells attained by them are listed below Leishmania Donovani infection:

seen as T shaped or double dot

Leishmania donovani (LD) bodies present both intracellularly (within the macrophages) and

extracellularly

• Haemophagocytosis : RBCs are seen engulfed by activated macrophages. Cells appear as stuffed

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Uses

Myeloperoxidase stain

:gives brown granular staining and used to differentiate between myeloid (+) and lymphoblastic (-) leukaemias .

Sudan black :

The reaction product is black and granular with reactivity similar in to myeloperoxidase and is used to differentiate between myeloid (+) and lymphoblastic (-) leukaemias .

Chloracetate estrase

: The reaction product is bright red with positivity in granulocytic cells and mast cells .The activity appears as the myeloblasts mature to promyelocytes and myelocytes which stain strongly . It is therefore useful as a marker of cytoplasmic maturation in granulocytic leukaemias with heavy cytoplasmic staining in acute promyelocytis leukaemia.

α naphthyl butyrate esterase

: The reaction product is brown and granular and is used to identify the monocytic population in AML M4 and M5

β naphthyl butyrate esterase :

gives diffuse red/brown staining and is used to identify the monocytic population in AML M4 and M5 .Less specific than α naphthyl butyrate esterase .

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Acid phosphatase:

Its reaction product is red and is used in diagnosis of T cell acute leukaemias and hairy cell leukaemia.

Periodic acid schiff:

The reaction product is magenta in colour. Two types of pattern are seen i.e. granular and diffuse positivity in granulocytic precursors & block positivity in lymphoblasts in acute lumphoblastic leukaemia

Toulidine blue:

The reaction is granular and bright red/purple in colour .It is used to highlight mast cells and basophils.

5. Hemosiderin.

Iron is essential for haemoglobin synthesis and is present in bone marrow as haemosiderin , which is the main storage form of iron in reticuloendothelial cells . .

Bone marrow iron stores : The iron stores (hemosiderin) can be assessed by using the Prussian (perl’s) blue stain .It is best performed on bone marrow aspirate smears.For grading of iron stores intracellular iron( present in macrophages with a small amount in erythroblasts) is evaluated as extracellular iron can be an artifact . The iron stores in bone marrow may be assessed as normal , decreased or increased and grading from 1+ to 6+ (as given below) is used .

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Bluish iron in Bone Marrow Aspirate smear (perl’s Prussian blue stain)

 Grades 1+ to 3+ are considered normal with decreased iron stores when < 1+ and increased iron stores when > 3+ .

Increased bone marrow iron  Anaemia of chronic disorder  Sideroblastic anaemias etc Decreased bone marrow iron

 Iron defeciency anaemia

The Prussian blue stain can also be used to identify sideroblasts which constitute 30-50% of erythroblasts in a normal marrow .These have small , few( 2-5), fine, scattered prussian blue positive granules in the cytoplasm . Another type of sideroblasts i.e. Ring sideroblasts seen as numerous and large granules ,frequently forming a complete or partial ring around nucleus can also be found in the marrow in conditions with disturbed haem synthesis

Ring sideroblasts are seen in  Sideroblastic anaemia  Lead intoxication  Megaloblastic anaemia

 Drug reaction : Chloramphenicol, Isoniazid etc.

Bone marrow biopsy interpretation

BMB is mandatory when aspirate is not obtained ("dry aspirate" or "dry tap"),the causes for which are listed below .

 Aplastic anaemia

 Bone marrow failure syndromes  Marrow fibrosis

 Marrow infiltration disorders(including metastatic deposits,lymphoma involvement )

 Hairy cell leukaemia  Granulomatous disorders  Pyrexia of unknown origin

Bone Marrow Biopsy has some advantages over aspirate as listed below :

 Examination of a greater volume of tissue with preserved architecture,  Assessment of cellularity

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 Detection of compact and/or fibrotic lesions,  Detection of metastatic deposits

 Identification of granulomas

 Application of immunohistochemistry (IHC).

Analysis of the BM biopsy : Bone marrow biopsy should be examined at minimum three levels .A systematic approach is needed which includes:

-Low power examination: evaluate for cellularity , megakaryocytes ,any focal lesions

-High power examination : evaluate for details of each cell line and detail of the focal lesion

i) Size / adequacy of the biopsy

 A bone marrow trephine biopsy specimen must be of adequate size to permit reliable interpretation of cellularity and to give a reasonable probability of detecting focal lesions.

 It should contain three or four intertrabecular spaces and be at least 0.5cm in length . The current recommendation for adequate biopsy being 1.6cm-2.0cm  Trephine biopsies composed of cortical bone and a small amount of

subcortical bone marrow are inadequate and may give a false impression of hypocellularity

a) Cellularity is assessed by roughly estimating the percentage of section occupied by the haematopoietic tissue in relation to the adipose tissue and this can vary with age.

Different patterns of cellularity in a young adult are as below :

Hypocellular Normocellular Hypercellular

The variations of cellularity with age have already being discussed in section on bone marrow aspiration

b) Megakaryocytes : these are the largest cells in marrow and are evaluated for distribution, number and morphology .

Distribution

- Normally the megakarycytes are perisinusoidal in central parts of marrow and are seen either singly or in clusters of ≤3 cells .

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-Myelodysplastic syndrome where the megakaryocytes are seen paratrabecular in location.

-Large clusters of megakaryocytes seen in myeloprolferative disorders and following chemotherapy.

Number : the causes of increased and decreased megakaryocyte number have already been discussed in section on bone marrow aspiration.

Morphology :

- Size : Large megakaryocytes in Essential thrombocythemia o Small megakaryocytes in Chronic melogenous leukaemia - Dysplasia : seen as anisocytosis, abnormal nucleocytoplasmic ratio ,

hyperlobulation and/or hypolubulated nuclei in Myelodysplastic syndromes and Chronic idiopathic myelofibrosis.

High power examination

For details of haemopoietic cell lines: ( distribution, maturation and morphology) Normal distribution

 Erythropoietic islands: perisinusoidal in central parts of marrow

 Myeloid cells : Precursors are mainly partrabecular in 2-3 rows .As the cells mature these lie in central parts of marrow cavity

 Other cells : show no particular pattern

a) Erythroid cells : present as clusters of erythroid cells in different stages of maturation seen around sinusoids .These cells are arranged closely adhered to each other and have round nuclei with condensed chromatin in late erythroblasts (can be confused with lymphoid cells which show coarse clumping of chromatin).These cells are evaluated for distribution ,number, maturation and morphology.

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 Distribution: Erythroid cells present in paratrabecular location in myelodysplastic syndrome

 Number: The causes of increase and decrease in erythroid cell number in marrow have already been discussed in section on bone marrow aspiration.

 Maturation and morphology :

-In regenerating marrow the islands of erythroid cells are composed of cells in same stage of maturation i.e. clusters of immature elements seen

-Dyserythropoiesis : as seen on bone marrow aspirates ( already discussed before )

b) Myeloid cells : In a normal marrow precursors of myeloid cells are mainly partrabecular in 2-3 rows and as the cells mature these lie in central parts of marrow cavity.

o Distribution abnormal in

 Myelodysplastic syndrome : Abnormal localisation of Immature Precursors(ALIP) : 3 or > foci of small aggregates of myeloblasts ,promyelocytes localised in central portion of marrow away from vascular structures and endosteal surface of bone trabecula .

 Myeloproliferative disorder (CML) : Paratrabecular cuff of immature neutrophils increased to 5-10 cells thick.

o Morphology : dysplastic changes as already discussed before under bone marrow aspirate examination.

c) Lymphoid cells : these show no particular pattern of distribution and are seen scattered in between the other haematopoietic elements .

 Increase in lymphoid cells : can be seen as a part of reactive process or a neoplastic proliferation .

 Arrangement of lymphoid cells :

 Reactive process may show interstitial arrangement or a nodular pattern . Interstitial is described as lymphoid cells interspersed between the normal haematopoietic cells with increase in number of lymphoid cells .The confirmation of nonneoplastic nature of lesion is done on immunohistochemistry Nodules of reactive lymphoid cells are small with well defined margins and composed of polymorphous population of lymphoid cells depicting different stages of maturation .

 Neoplastic proliferation oon the other hand shows three types of pattern:

 Interstitial  Nodular  Diffuse

(27)

Nodular pattern : seen as large nodules with ill defined margins often extending around fat cells and composed of homogenous population of lymphoid cells .

Diffuse pattern: replacement of normal marrow( distorting archtitecture of marrow ) by diffuse sheets of neoplastic lymphoid cells .

d) Abnormal cells: which are identified as: - Blasts of different cell lineage - Lymphoma infiltration by marrow - Myeloma cells

- Metastatic deposits

Note: For details of individual cell types kindly refer to standard text books.

Biopsy showing sheets of blasts –acute leukaemia

(28)

Biopsy of Multiple myeloma with shets of plasma cells

Granulomas (arrows) in bone marrow

Oxalate crystals with gaint cell raction in patient with oxalosis

Metastatic deposits : seen as clusters and glandular arrangement of hyperchromatic & pleomorphic cells

(29)

Storage disorder : Gaucher cells with abundant tissue paper cytoplasm

Quantification of BM reticulin

Reticulin is an early form of collagen( connective tissue) which is always present in small amount in normal bone marrow i.e. mainly perivascular and periendosteal in location. Reticulin can be stained using silver impregnation technique or Massons trichrome stain (stains collagen).The silver impregnation technique is routinely used in many laboratories and it stains reticulin black .

Reticulin deposition can be graded as follows

The majority of normal subjects have grade of 0-1.While grading reticulin the perivascular and periendosteal areas should be disregarded as these may give a false high grade .

Normal reticulin Increased reticulin

Causes of Increased reticulin

 Idiopathic myelofibrosis  Secondary myelofibrosis

o Myeloproliferative disoreders o Hodgkin’s disease

(30)

o SLE o AIDS

Bone marrow artifacts

Various artifacts in bone marrow aspirate and biopsies need to be recognized so that they are not misinterpreted. These artifacts can be introduced during the procedure or while processing in the laboratory.

Bone marrow aspirate artifacts :

1. Procedure :

- Inadequate particles: because of poor technique

- Abnormal spreading: cell crushing and distortion because of excess pressure applied.

- Clotted specimen

2. Processing:

- Inadequate drying of the film: the nuclear contents are seen leaking into the cytoplasm and cellular outline is indistinct.

- Poor fixation: because of water uptake into methanol used for fixation .It causes refractile inclusions in the red cells and poor definition of cellular details.

- Delayed fixation and staining which gives strong blue tint to the film

3.

Extraneous material

: like crystals of glove powder which stains blue with May Grunwald Giemsa stain and red with Periodic acid Schiff stain .

Crush artifact because of abnormal spreading Artifact because of inadequate drying of film

Bone marrow biopsy artifacts

1. Procedure :

- Too short biopsy sampling

- Mainly subcortical bone in biopsy sample when needle introduced at wrong angle : falsely hypocellular specimen

- Use of blunt needle leads of crumbling of bone to amorphous material - Torsion artifact elongated nuclei produced by twisting of the needle - Aspiration artifact : when aspiration performed immediately before a

(31)

2. Processing :

- Poor fixation : glassy nuclei with detailed structure cannot be recognized

- Shrinkage artifact : a clear space seen around the erythroblasts and megakaryocytes - Poor stainig : excessive decalcification

3. Extraneous material

Special tests on bone marrow

Immunophenotyping

Detection of antigens on the cells by using monoclonal antibodies .

Techniques utilized :

Flow cytometry: when the cells bearing the antigen are identified by means of monoclonal antibodies labeled with a flourochrome .Peripheral blood sample ( when significant number of abnormal cells are present) and bone marrow samples are used.

Immunohistochemistry or immunocytochemistry : when the cells bearing the antigen are identified by means of monoclonal antibodies applied to fixed cells or tissue sections on glass slides .Peripheral blood sample ( when significant number of abnormal cells are present) , bone marrow samples or a washed mononuclear cells isolated from blood or bone marrow are used.

Used in

- Diagnosis and classification of haematological neoplasms - For detection of minimal residual disease.

(32)

Refers to study of chromosomes and their abnormalities.

Chromosomal abnormalities are a common finding in haematological neoplasms so much so that some neoplasms are defined more precisely by the percentage of specific chromosomal abnormalities like acute myeloid leukaemias with recurrent genetic abnormalities

Used for :

1. Confirmation of diagnosis e.g. by demonstration of t (15;17) in M3 variant of AML

2. Also certain chromosomal abnormalities offer prognostic information e.g. in acute myeloid leukaemias presence of t(8;21) ,t(15;17) or inv (16) are indicative of better prognosis.

3. Confirmation of neoplastic processe

4. Monitoring the treatment like in Chronic myelogenous leukaemia by estimation of Philadelphia positive [ t (9: 22) ]metaphases .

Performed on cell suspensions obtained from peripheral blood or bone marrow .