Bone marrow Indication and interpretation

Speaker: Subhajit Hajra

Moderator: Dr. Tapan Kumar Ghosh

Anatomy..

Subsequent sites of Hematopoiesis in fetal life

• Throughout fetal life, the liver is the chief organ for production of myeloid and erythroid cells

After birth, BONE MARROW becomes main hematopoietic organ.

Pattern of distribution..• At birth, all bone marrow is red. • With age, more and more of it is

converted to the yellow type; only around half of adult bone marrow is red

• In Adults Red marrow is found mainly in the Flat bones and in the   epiphyseal ends of long bones such as the femur and humerus

Bone Marrow• Bone marrow is specially designed to support the

proliferation, differentiation, and maintenance of hematopoietic cells

• The stroma of the bone marrow is all tissue not directly involved in the primary function of hematopoiesis.

• Cells that constitute the bone marrow stroma are:• fibroblasts (reticular connective tissue)• macrophages• adipocytes• osteoblasts• osteoclasts• endothelial cells, which form the sinusoids.

How the test is performed…• Posterior iliac crest.• However, an aspirate can

also be obtained from the sternum (breastbone)--Contraindicted in patients with lytic bony lesion

• A trephine biopsy should never be performed on the sternum, however, due to the risk of injury to blood vessels, lungs or the heart.

• medial side of tibia upto 18 months of age

• Anterior illiac crest

• Salah's Bone Marrow Needle, iliac crest, with adjustable stop

• 14 (2.0mm) x 50mm• 16 (1.6mm) x 50mm• 18 (1.2mm) x 50mm (in child)

•  The aspirate yields semi-liquid bone marrow, which can be examined • under a light microscope as well as• analyzed by flow cytometry,•  chromosome analysis, or•  polymerase chain reaction (PCR).

• Frequently, a trephine biopsy is also obtained, which yields a narrow, cylindrically shaped solid piece of bone marrow which is examined microscopically (sometimes with the aid of immunohistochemistry) for cellularity and infiltrative processes.

Indication•Investigation of unexplained anemia ,

abnormal red cell indices, cytopenias or cytoses.

•Investigation of abnormal peripheral blood smear suggestive of bone marrow pathology,

•Diagnosis , staging and follow up of maligant haematological disorders.

Indication•Investigation of suspected bone marrow

metastases

•Unexplained focal bony lesions on radiological imaging

•Unexplained organomegaly or presence of mass lesions unavailable for biopsy

Indication•Microbiological culture for pyrexia of

unknown origin or specific infections , eg. Miliary tuberculosis , leishmaniasis , malaria

•Evaluation of iron stores

•Investigation of lipid/glycogen storage disorders

Indicaion•Exclusion of hematological disease in

potential allogenic stem cell transplant donors

•HPS•Plasma cell dyscrasias

Contraindications

• The only absolute reason to avoid is the presence of a severe bleeding disorder

Complications• While mild soreness lasting 12-24 hours is

common after a bone marrow examination, serious complications are extremely rare.

Erythroid seriesMyeloid seriesMegakaryocytic seriesMonocytic series

*Proerythroblast**Early erythroblast (Basophillic )***Intermediate erythroblast (Polychromatic)****Late erythroblast (Orthochromatic )

proerythroblast• Normal

proerythroblast [dark red arrow] in the bone marrow. This is a large cell with a round nucleus and a finely stippled chromatin pattern. Nucleoli are sometimes apparent.

• The cytoplasm is moderately to strongly basophilic.

Basophilic erythroblast • Spherical nucleus,

nucleoli not visible, basophilic cytoplasm

Polychromatophilic erythroblast• smaller nucleus –

condensed chromatin, baso- and eosinophilia in the cytoplasm

Orthochromatophilic erythroblast • small nucleus with highly

condensed chromatin, nucleus extruded, eosinophilic cytoplasm

Normal erythroblasts in the BM

credit-Dr. Lekstrom Hines, J EM 1999

Normal granulocyte precursors in the bone marrow

• Note the myeloblast [dark red arrow] with a high nucleocytoplasmic ratio, diffuse chromatin pattern and nucleolus.

• There is a promyelocyte [green arrow] which is larger and has a lower nucleocytoplasmic ratio and abundant azurophilic granules.

Myelocytes • Myelocytes are

smaller than promyelocytes and have specific granules that indicate whether they are of neutrophil, eosinophil or basophil lineage.

• The nucleolus is no longer visible.

Eosinophilic myelocyte

A neutrophil metamyelocyte•The metamyelocyte

differs from a myelocyte in having some indentation of the nucleus

• It differs from a band form in not having any part of its nucleus with two parallel edges

Neutrophilic metamyelocyte

Eosinophilic metamyelocyte

Basophilic metamyelocyte

Band or juvenile Neutrophils

•There are smaller numbers of cells of neutrophil lineage with non-segmented nuclei. They are referred to as neutrophil band cells or band forms. They are less mature than segmented neutrophils.

.

*Megakaryoblasts** Promegakaryocytes***Megakaryocytes

Megakaryoblasts •Megakaryoblasts

are the precursors of the megakarycytes.

•They may show cytoplasmic blebbing.

Promegakaryocyte

Megakaryocyte

Monocytopoiesis

- Monoblast

- Promonocyte

- Monocyte

Promonocyte

Scheme for evaluation of bone marrow•Clinical History, cliniacal data,PBS•Amount•Fixed for 15 mins and stained •blood clot present in the needle or slide

may be processed and H/P examination may reveal metastatic deposit,multiple myeloma etc.

•Smear should be first looked at scanner to identify bone marrow particles.

Systemic scheme for Examining aspirated BM films•Low power (x10)

▫Determine cellularity▫Identify megakaryocytes▫Look for clumps of abnormal cells▫Identify macrophages,reticulum cells▫Bone Marrow stroma▫Osteoclatic giant cells

Scheme for Evaluation aspirated BM filmsHigher power (x40, x100)

▫Cells of erythroid ,Myeloid series ,plasma cells,osteoblasts ,osteoclastic giants cells

▫Identify all stages of maturation of myeloid and erythroid cells.

▫Determine the M:E ratio▫Perform a differential count--atleast 500

cells▫Look for areas of BM necrosis.▫Assess the iron content.

Assessment of BM cellularity• Cellularity cannot be assessed without

knowing the age of a patient.• A young child on average has about 80%

of the intertrabecular space occupied by haemopoietic cells whereas in a 75-year-old the average has fallen to around 30%.

•Choose the marrow with particles--examining single marrow particle may lead to spurious results

•Examine atleast 3-4 slides•100 – Age of Patient

Comparing normo, hyper, & hypocellular marrows

Asessment of bone marrow cellularity•Dry tap: Aplastic anaemia,hypoplastic MDS

(5%),hypoplastic leukemia,subcotical fatty marrow,fibrosis --AML M7,long standing myeloproliferative disorder,hairy cell leukemia,multiple myeloma , metastasis

•Marrow failure states--fanconi's anaemia, PNH, hypoplastic MDS,Diamond Blackfan syndrome, Schawman Diamond sydrome, Dyskeratosis congenita

Asessment of bone marrow cellularity•Disadvantages:•solid metastatic tumors, focal lesions like

granuloma, multiple myeloma may be missed

•subcortical aspiration may lead to false reporting of hypoplastic anemia

The nucleated differential count•To assess hematopoietic activity and

compare the proportion of different cell lineage with known reference range

•Quantify abnormal cells•Cell trails behind the marrow particles as

these are least dilutated with blood ,close represntative of marrow particle,less smudge cells and dispersed with good cytological detail

The nucleated differential count•Blasts,Cells of granulocytic, monocytic

series,lymphocytes and plasma cell and erythoblasts are included in NDC

•Megakaryocytes, stromal cells, osteoblasts, osteoclasts, metastatic cell, smudge cells are excluded from NDC

•Lymphoid follicles if present should not be included in NDC but should be commented upon.

M:E ratio•The M:E ratio is the ratio of all

granulocytic plus monocytic cells (Myeloid) to all erythroblasts (Erythroid).

•For all bone marrow aspirates examined, the report should specify the M:E ratio and the percentage of lymphocytes and plasma cells.

•A differential count of at 500 cells should be performed.

•M:E ratio of 3;1 -15:1 is considred normal

Bone marrow iron stores• Once all normal and

abnormal bone marrow cells have been assessed on a routine stain an iron stain should be examined, using a medium power objective (X 40 or X 50). Storage iron, which stains blue in prussian blue should be assessed in bone marrow fragments. This image shows normal bone marrow iron.

Bone marrow iron stores•Evaluation of stored iron and sideroblasts•A bone marrow smear with increased iron

content should be taken as positive control•Iron staining is mandatory for intial work up

of bone marrow aspirate but may be excluded in follow up cases of acute leukemia

•Particle clots are also can be stained for iron•Biopsy is less reliable for measuring iron

stores than bone marrow aspirate

Bone marrow iron stores•Iron stores are evaluated examining bone

marrow macrophages/reticulum cells examining several marrow particles

•Iron stores may be graded as follows 0- No stainable iron 1+- Samll iron particles just visible in

reticulum cell using 100x 2+-Small sparse iron particles in

reticulum cells visible at 40x

Bone Marrow iron stores 3+-Numerous small particles in reticulum

cells 4+- Larger particles with a tendency to

aggregate in clumps 5+- Dense large clumps 6+- Very large clumps and extra cellular iron•Quantitative estimation of iron may be

subjected to inter observer variation and reports may not be reproducible

Bone marrow iron stores •No of sideroblast should be noted with

frequency and location( Cytoplasmic or perinuclear) of sideroblastic granules

•100 RBC are evaluated to find ring sideroblasts in a iron stained smears

Erythroid series•look for cellularity-- hypo,hyper or normo•Maturation:Early :Intermediate :Late

normoblast(1:2:4)•Reaction: Normo, Megalo, Micro

normoblastic•Dyserythropoiesis•Mitosis

to be continued..................