Bone Marrow Procedures and

ProcessingDr Richards Kakumanu

ModeratorsDr V Vijay Sreedhar (Prof & HOD)Dr Manimekhala (Assoc. Prof)

Upgraded Department of Pathology

Marrow Specimen

Needle Aspiration

Percutaneous Trephine Biopsy

Surgical Biopsy

Why do we need to do bone marrow studies?

Bone Marrow Aspiration

•Simple and safe

•Repeated many times

•Performed on outpatients

•Safe in almost circumstances

Advantages

•Individual cells are well preserved

•Subtle differences between the cells recognized

Disadvantages

•Arrangement of cells in the marrow

•Relationship between the cells

•Fibrotic marrows: aspiration of blood

General considerations

•Iliac spines: advantages of trephine biopsy

•Obese and immobile patients: technical difficulties

•Sternum should be avoided in children

•Danger of perforating inner cortical layer and damage to the underlying large blood vessels and right atrium

Important considerations

•Always wear surgical gloves

•Avoid needle stick injuries

•Local and oral analgesia

•Use only needles designed for the purpose

Marrow puncture needles

•Needles should be stout

•Hard stainless steel

•About 7-8 cm in length

•Well fitting stilette

•Adjustable guard

•Most common reusable needles: Klima and Salah

•Point of the needle and the edge of the bevel must be kept well sharpened

•Islam's bone marrow aspiration needle: the dome-shaped handle and the T-bar are intended to provide stability and control during the operation

•Disposable bone marrow aspiration needles

•Clean the skin

•70% alcohol

•0.5% chlorhexidine(5% diluted 1 in 10 in ethanol)

•Infiltrate the skin, subcutaneous tissue, and periosteum

•2% lignocaine 2-5ml

WAIT...

•Pass the needle with a boring movement

•Needle - perpendicular into the cavity

•Remove the stilette when bone has been penetrated

•Attach a 1-2ml syringe

•Aspirate marrow contents

•Second sample: Attach a second 5-10 ml syringe for cytogenetic and immunophenotypic analysis

•As a rule material can be sucked into the syringe without difficulty

•If unable to aspirate: Insert the stilette, push the needle and then Aspirate

•Dry tap: failure to Aspirate marrow suggests fibrosis or infiltration

•Dry tap: insert the stilette, push any material in the lumen onto a slide

•Obese patients: CT guided marrow sampling

•Ethylenediaminetetra-acetic acid(EDTA)

•Preservative free heparin: phenotyping and cytogenetic analysis

•Preservative in fixative: histopathology

•Fixation in absolute methanol: romanosky method or perls' stain or cytochemical staining

Puncture of the Ilium

Puncture of the ilium•Usual sites: Posterior and anterior iliac spines

•Center of the oval posterior superior iliac spine

•2cm posterior and 2cm inferior to anterior superior iliac spine

•Posterior iliac spine: 1) overlies a large marrow containing area. 2)relatively large volumes of marrow can be aspirated

•Posture: 1)patient lying sideways or 2)prone

•Anterior superior iliac spine: 1) overlying bone is thinner than iliac crest 2) very obese

Puncture of the sternum

Puncture of the sternum

•Avoid pushing the aspiration needle through the bone

•Usual sites: 1) manubrium, 2) 1st or 2nd parts of the body of sternum

Manubrium•Denser bone than body of

sternum

•More fatty marrow in elderly subjects

•Thickness of cortex: 0.2-5.0mm

•Difficult to ascertain that the needle point has reached the cavity of the bone

•Site: about 1cm above the sternomanubrial angle and slightly to one side of the midline

Body of Sternum•Site: opposite the second

intercostal space

•Slightly to one side of the midline

•Essential: Needle with a guard

•Adjust the guard When needle reaches the periosteum

•Allow it to penetrate about 5mm further

•Push the needle with a boring motion, enter the cavity and Aspirate

Puncture of the Spinous process

Puncture of the Spinous process•Done in adults

•Spines of the lumbar vertebrae

•Not difficult as bones lie superficially

•More pressure is required

•Patient sitting up or lying sideways

•Pass the needle at right angles to the skin surface

•Into the spine of the Lumbar vertebra slightly lateral to the midline

Comparison of different sites for marrow puncture •In general, the overall cellularity, the hemopoietic

maturation pathways, and the balance between erythropoiesis and leucopoiesis are similar at all sites

•Considerable variation in the composition of the cellular marrow in certain conditions

•Aspiration from only one site may give misleading information. Eg: aplastic anemia - patchily affected

•Dry tap/bloody tap: advantage in choice of several sites

Aspiration of bone marrow in children•Small babies: medial aspect of

upper end of tibia, just below the level of tibial tubercle

•Caution! Vulnerable to fractures and laceration of adjacent major blood vessels

•Children: iliac puncture(posterior spine)

•Older obese children: anterior ilium

•Tibial cortical bone: too dense, marrow normally less active

•Sternal puncture avoided in children

Processing of Aspirated Bone Marrow

•Preparing films from Bone Marrow Aspirates

•Concentration of Bone marrow by centrifugation

•Preparation of films of post-mortem bone marrow

Processing of marrow Aspirates

General considerations•0.3 ml of marrow fluid from a single site

•>0.3ml: little advantage - peripheral blood dilution

•A second syringe: 5-10 ml of marrow - immunophenotyping, cytogenetics and molecular studies

•Sample of peripheral blood: finger prick or venepuncture

•Good practice: obtain full blood count and storage

•The blood film should be permanently stored with the bone marrow films

Preparing films from Bone Marrow Aspirates•Smears should be made without

delay

•Smear length: 3-5cm

•Glass spreader: smooth edged, not more than 2cm width

•Marrow fragments dragged behind the spreader

•Fragments leave a trail of cells behind them

•Spreading should be towards the label area.

•Avoids particles dragged to the tip of the slide

•Insufficient fragments: can be concentrated

•Deliver single drops of Aspirate onto slides about 1cm from one end

•Most of the blood is quickly sucked off from the edge of the drop using the marrow syringe or a fine plastic pipette

•Irregularly shaped marrow fragments tend to be left behind, can be lifted off with a spreader

•Smears can be made

•Thorough drying, fix the smears, and stain (Romanosky)

•A longer fixation time(at least 20 min in methanol) is essential for high-quality staining

•Perls' method: demonstrates the presence or absence of iron

•Atleast one film should be fixed for perls' stain

•Overnight drying may be necessary to achieve to achieve optimal results

•Satisfactory: only when marrow particles and free marrow cells can be seen in stained films

•Differential counts should be made in cellular trails commencing from the marrow fragment and working back towards the head of the film

•Smaller numbers of cells from the peripheral blood are included in a differential count

•Appropriate amounts of anticoagulant for the volume of marrow to be anticoagulated are used

•Gross excess of anticoagulant: masses of pink-staining amorphous material may be seen

•Clumping of some erythroblasts and reticulocytes may be seen

Concentration of bone marrow by centrifugation

•To concentrate the marrow cells

•To assess the relative proportions of marrow cells, peripheral blood and fat in aspirated material

•Useful in poorly cellular samples

Preparation of films of post-mortem bone marrow •Smears: Seldom satisfactory

•Satisfactory results: procedure must be Carried out as soon after death as possible

•Majority of cells tend to break up when making films

•Better preservation: a small piece of marrow is suspended in 1-2ml of 5% bovine albumin( 1 vol. 30% albumin, 5 vol. 9 g/l NaCl)

•The suspension is then centrifuged

•The deposited marrow cells are resuspended In a vol. of supernatant approx equal to or slightly less than that of the deposit

•Films made from the suspension in the usual way

Trephine Biopsy

•A little less simple than aspiration

•Can be performed on outpatients or at bedside

•Structure of relatively large pieces of marrow

•Imprint smears: morphological features of individual cells

•Superior for diagnosing marrow involvement by lymphoma and non hematological neoplasms

•Invaluable in the diagnosis of conditions that yield a "dry tap".

•Hodgkin's disease, lymphoma: disrupted architecture of the marrow is an important diagnostic feature

•Usual site: posterior iliac spine

•Posterior iliac spine: longer, larger samples. Less comfortable for the patient

•Anterior iliac spine can also be used

•Insert the biopsy needle into the bone

•Obtain a core of tissue using a to-and-fro rotation

•Main problems: specimen may be crushed, distortion of the architecture, difficulty to detach the core of the bone from inside the marrow space

•Trephine biopsy needles: specifically designed to overcome theses problems

Jamshidi trephine•Tapering end to reduce crush

artefact

Islam trephine•Has a core securing device

•The distal cutting edge is shaped to hold the core secure during extraction of the material

•Larger specimens: trephine needles with bores of 4-5mm

•Occasionally used needles: 2-mm bore microtrephine, Vim-Silverman needle

•Smaller yield of marrow specimen that are prone for fracturing

Thrombocytopenia and neutropenia in small preterm neonates•19 G, half-inch Osgood needle

•Introduced 2cm below tibial tuberosity

•The trocar is removed

•The hollow needle is advanced by twisting 2-3mm into the marrow space

•Suction applied with a syringe until marrow appears

•Then needle and syringe are withdrawn

•The marrow clot is gently dislodged with the tip of a needle and placed into fixative

•The specimen is processed

•Decalcification is not required

Complications of bone marrow biopsy

•Generally a safe procedure

•Serious adverse events <0.05% of procedures

•Most common complication: bleeding

•Gluteal compartment syndrome

•Very rarely death

•Bleeding is related to impairment of platelet function than to thrombocytopenia or a coagulation factor defect

Imprints from bone marrow trephine biopsy

•Can be taken before the specimen is transferred into fixative

•Particularly useful if the bone marrow Aspirate is inadequate

•Bony core is gently dabbed or rolled across the slide

•Fixed and stained

•Allows immediate examination of cells that fall out of the specimen onto the slide

•May provide a diagnosis several days before trephine biopsy has been processed

Processing of bone marrow trephine biopsy specimens

•Fixed in 10% formal saline

•Buffered to ph 7, for 12-48 hrs

•Decalcifying, dehydrating and embedding in paraffin wax

•Cell shrinkage and distortion from the decalcification process may distort cellular detail

•Methyl methacrylate(plastic) embedding

Staining of Sections of Bone Marrow Trephine Biopsy Specimens

•Routinely stained for H& E

•Silver impregnation method for reticulin

•Romanosky dyes: MGG - hemopoietic cells may be more easily identified

•Perls' reaction for iron

•H&E staining: excellent for demonstrating the cellularity and pattern of the marrow

•Pathological changes: fibrosis, granulomata, carcinoma cells

•IHC: paraffin/plastic embedded specimens

•Silver impregnation: stains the glycoprotein matrix

•Reticulin: an early form of collagen

•Increase in marrow reticulin: increase in the number and thickness of fibers

•Eg: myeloproliferative disorders - proliferation of megakaryocytes, lymphoproliferative disorders, secondary carcinoma with marrow infiltration. Osseous disorders such as hyperparathyroidism and paget's disease and inflammatory reactions.

•In myelofibrosis or myelosclerosis, a more "mature" form of collagen is present

•Visible on H&E staining

References

•Dacie and Lewis practical hematology

•Bone Marrow Pathology - Barbara Bain - 4th edition

•Wintrobe's clinical hematology

•Google images

Thank you