Bone Marrow Aspiration and Biopsy: Bone marrow aspiration means to withdraw the fluid portion of the marrow and the bone marrow biopsy means to take out a piece of bone core with help of a wide bore needle. Bone marrow aspiration and biopsy are collectively referred to bone marrow examination.
Bone marrow drilling is one of the oldest surgical practices which still have relevance and is most basic and important test in the field of medicine. The currently practised way of testing bone marrow came into practice since 1905 after a report by “Pianese” of marrow Leishmaniasis. In modern medicine, bone marrow examination is a valuable tool in evaluation of varied diseases.
INDICATION OF BONE MARROW ASPIRATION AND BIOPSY
• Diagnosis, staging and monitoring of therapeutic response in various hematological diseases like:
– Acute leukaemias
– Lymphoproliferative disorders
– Myeloproliferative disorders
– Myelodysplastic syndrome
– Plasma cell disorders (multiple myeloma, primary amyloidosis, etc.)
• To rule out metastasis
• Evaluation of cytopenias, unexplained anaemia, leukocytosis, thrombocytosis, erythrocytosis
• Evaluation of iron status
• Pyrexia of unknown origin
• Chronic and disseminated infections and granulomatous diseases like tuberculosis, leishmaniasis, fungal infection, sarcoidosis, HIV specially when associated with cytopenias
• Unexplained splenomegaly
• Storage disorders (Gaucher’s disease, etc.)
• Chromosomal disorders in neonates.
CONTRAINDICATION OF BONE MARROW ASPIRATION AND BIOPSY
There is actually no absolute contraindication. All of the mentioned below are relative contraindications.1,2 It is imperative to ensure hemostasis after the procedure especially in a patient with coagulopathy or thrombocytopenia.
• Hemophilia
• Disseminated intravascular coagulation (DIC) or severe bleeding disorder(coagulopathy)
• Severe thrombocytopenia
• Avoidsternal aspirate in suspected multiple myeloma (high-risk of perforation)
• Avoid in bone suspicious of osteomyelitis.
ANATOMY OF BONE MARROW ASPIRATION AND BIOPSY
Bone marrow is a spongy tissue which consists of hematopoietic stem cells along with maturing hematopoietic elements, supporting stroma (macro- and microenvironment) and fat. The tissue lies within the hollow center of all the bones. At birth, almost all the marrow spaces are filled with hematopoietic tissue. As age advance, the tissue gets replaced by fat in long bones so in an adolescent and adults, the hematopoietic tissue is chiefly confined to axial skeletal (flat bones). Not only the bone marrow shrink in its extent but also the cellular portion decreases in whatever the bone marrow remains there in adults with advancing age. Whereas a marrow space shows a nearly 100% areas containing cellular components in a new born, the adults have it in range of 30–70% depending upon age.
TECHNIQUE AND EQUIPMENT OF BONE MARROW ASPIRATION AND BIOPSY
Site Selection
• Posterior superior iliac spine (PSIS) is the most preferred site due to several reasons. Apart from advantage of having a good amount of marrow tissue in iliac crest, the PSIS is a superficial and fairly immovable landmark which can be palpated and located easily. The thickness of cortical bone is lesser at PSISas compared to other parts of iliac crest. The thickness of iliac crest blade at or near PSIS is more so that we can get a good length marrow biopsy and the chance of cutting through the bone is less. Moreover, the amount of pain felt is usually lesser than any other site in body.
• Anterior superior iliac spine (ASIS) and area 2.5–5 cm posterior to it can be chosen as second option in a person who is not able to lie on lateral positions or having a disease/deformity affecting PSIS. ASIS can be accessed in supine posture. ASIS is also easily palpable and a relatively fixed landmark. The Skin overlying ASIS is not fixed and can slide during the procedure making it difficult to poke the bone at desired site. The thickness of cortical bone is more at this site so, it sometimes becomes difficult to get access to marrow spaces.
• Sternum is last option and should be avoided in routine. Sternal puncture should never be done in patients younger than 12 years. Sternum can be accessible in morbidly obese patients. The preferred site is area close to second or third intercostal space. A bone marrow biopsy should never be attempted and only bone marrow aspiration can be done.
• Tibia contains marrow in infants and can be a good site for bone marrow sampling in infants. Ideal site is medial surface of tibia just below tibial tuberosity. Both marrow aspiration and biopsy can be done from this site.
• Surgical biopsy (open biopsy) or CT-guided biopsy (ribs, vertebrae or greater trochanter) can be done to target the affected sites of bone/bone marrow. Such kind of procedures is useful in diseases with focal bone marrow involvement. Bone Marrow Aspirate, Smear and Imprints/Touch Smears are good for morphologic assessment of cells and to get a differential counts (myelogram). If marrow fragments are visible then an idea of the cellularity of the marrow can also be taken. Occasionally, the marrow aspirate is diluted with blood and no or only occasional marrow components are visible. In such cases, the diagnosis rests on marrow biopsy. The imprint/touch smears made by putting and rolling of bone marrow biopsy piece on slides can help to get morphologic details of cells in such cases. Apart from routine morphological tests on marrow, the slides can be tested with various cytochemical stains to look for particular type of disease for example, the myeloperoxidase (MPO) stain for acute myeloid leukaemia and iron stain to look for iron stores and also to diagnose myelodysplastic syndromes. The aspirated marrow can be taken into heparinized or ethylenediaminetetraacetic acid (EDTA) vials to subject to various molecular/fluorescence in situ hybridization (FISH), cytogenetic studies and flow cytometric immunophenotyping. Other tests which can be performed at aspirated marrow are cultures for mycobacterium, Leishmania, bacteria/fungi, etc. as indicated.
Bone Marrow Biopsy
Biopsy is a long thin piece from core of the bone. We should try to get at least 1.5 cm long piece so that to see at least 10 marrow spaces (intertrabecular spaces) under the microscope to call it an adequate biopsy, otherwise you may get an equivocal report from your pathologist or the pathologist can state that biopsy was inadequate to give a final opinion. Biopsy is good tool to assess cellularity of marrow and arrangement or localization of various cellular components within the marrow spaces. Biopsy is also useful to look for malignant infiltrates like solid tumors, lymphomas. Biopsy from bilateral PSIS or iliac crests is sometimes recommended to more reliably look for lymphoma or solid tumor infiltrates. Nowadays, bilateral biopsies are not routinely recommended and in diseases with known focal marrow involvement, it is preferable to do computed tomography (CT) or positron emission tomography (FDG-PET) guided biopsies. Other tests which can be performed at biopsy tissue are reticulin stain for fibrosis and immunohistochemistry as indicated.
Equipment
• Bone marrow aspiration needle: Usually a smaller, 16-gauge needle with a stylet
• Bone marrow biopsy needle: A wider bore, usually 11 gauge for adults (some prefer 9-gauge needle) or 13-gauge needle for pediatric age group. Needle is accompanied by a stylet and a “J” shaped device (obturator) for removing the biopsy core from the needle
PREPARATION OF BONE MARROW ASPIRATION AND BIOPSY
• Bone marrow aspiration and biopsy should be an absolutely aseptic procedure. Every care should be taken to maintain so, to avoid any chances of infection
• Consent: A written informed consent should also be signed, explaining the procedure and the possible risks involved in it. This also leads to allay the apprehension in the patient’s mind and make him feel comfortable
• General anesthesia or sedation may be required in pediatric cases or in anxious/non-cooperating adults
• Monitoring required: Usually, the vitals and oxygen saturation are checked before starting the procedure. A check is also kept on the oxygen saturation in cases of mild sedation
• Laboratory work-up: Generally, coagulation profile and platelet counts are checked before the procedure
• Arrange major equipment (bone marrow aspiration and biopsy needles of appropriate size
• Arrange adjunct equipment:
– A thin knife (usually size 11) to give a stab at the selected site
– Five disposable syringes with needles, 5 and 10 cc (to give local anesthesia and then to collect samples)
– Glass slides (should be a fresh pack or recently opened pack)
– Glass or plastic container with 10% formalin or B5 solution to put biopsy in it
– Heparinized/EDTA vials, culture bottles, etc. as per the requirement
– Cleansing solutions (alcohol and povidone-iodine or 2% chlorhexidine),sterile pack containing bowls, drapes, sponge holder, gauge pieces/pads, injectable lidocaine 2% for local anesthesia
• Also, one of the most important is an assistant who is good in making slides.
PROCEDURE OF BONE MARROW ASPIRATION AND BIOPSY
For Posterior Iliac Crest Site
• The patient should be placed in lateral decubitus position (if you are a right handed person then left lateral position will be more convenient) with the top leg flexed and the lower leg straight. Alternatively, prone positioning can be done.
• Examine site for any evidence of infection; palpate and locate the posterior iliac crest and PSIS. ASIS should also be palpated and located, as the needle will be pointed in this direction once the bone has been entered.
• Common site for aspiration and biopsy is approximately three finger-widths from the midline and two finger-widths inferior to the iliac crest.
• Using sterile technique, protective clothing and gloves (and eye wear if necessary), the bone marrow tray should be first opened and organized for easy access to needed items. Needles, stylets, and plastic syringes should be checked to ensure that they are intact and function properly.
• Cleanse the chosen area with povidone-iodine or chlorhexidine solution and drape a sterile field.
• Use 1–2% lidocaine solution using a 23-gauge needle to anesthetize the skin, subcutaneous tissue. Infiltrate the perisotium repeatedly by injecting small amounts of lidocaine solution at different points on the surface of the bone with a 21–22-gauge needle. Anesthetize a small area around the target region as aspirate and biopsy should be taken from different sites.
• While waiting forthe anaesthetic to produce its effect, extra syringes for special studies (e.g. flow cytometry, cytogenetics and molecular studies) can be appropriately anticoagulated. Specimens for molecular studies should not contain heparin.
• Once local anaesthesia has been achieved, make a small (3 mm) skin incision with a scalpel blade at the site of insertion of the aspiration needle. The incision should be perpendicular to the long axis of body to avoid gaping of wound.
• Hold the bone marrow needle (with stylet in place) perpendicular to the skin at the previously marked point, and gently advance it to the periosteum. When the needle has reached periosteum, it should be directed toward the ASIS.
• Use twisting motion and do not twist more than 180 degree in either direction to penetrate the periosteum. A steady twisting back and forth motion. When the periosteum is penetrated there is a give way feeling. At this point patient feels deep seated pain. Patient should be alerted of this pain beforehand otherwise he may be taken by surprise. Continue to advance the needles lightly to ensure that it is anchored into the bone.
• Remove the stylet, attach a 2 mL syringe to the aspiration needle, and again advise the patient that the aspiration may cause a brief period of pain.
• Only aspirate 0.2–0.5 mL of bone marrow as excessive drainage may lead to dilution with peripheral blood. This initial specimen should be used for preparing smears.
• The non-anticoagulated specimen should be handed to the assistant, who will assess the quality of the sample (i.e. determine the presence or absence of grossly visible bone spicules) and prepare the various smears. Anti-coagulated specimens should be sent to the laboratory for further preparation and other tests (e.g. cytogenetics, molecular studies, cultures and flow cytometry). The patient should be made aware of the need for multiple specimens at the outset, since each separate aspiration may be painful, despite fully adequate local anesthesia.
• If aspiration attempts are not successful,reinsert the stylet(the needle may be rotated) and advance theneedle a short distance;repeat attempts at aspiration with the syringe and suction. If multiple aspiration attempts are unsuccessful, an alternate site (e.g. the other posterior iliac crest) may be approached with the same sterile strategy after the bone marrow biopsy has been obtained.
• Once it has been determined that the aspirate is satisfactory, reinsert the stylet and remove the needle (with stylet in place) by using a similar twisting motion, and apply pressure to the site with small gauze until the bleeding stops.
• If a biopsy is necessary, prepare the Jamshidi™ needle and advance it into the cortical bone, using the same incision but a slightly different point in bone, with a steady twisting movement until it is firmly lodged. This may require a greater amount of pressure than was used forthe aspiration. Remove the stylet and with a rotating motion advance the needle for another 15–20 mm.
• Redirect the needle tip and rotate it 360 degrees in both directions to separate the biopsy specimen from the surrounding marrow tissue. Following this step, the needle should be advanced a very short distance prior to removal. This step may prevent the specimen from being pulled out of the needle at the biopsy site.
• Remove the needle with a slight twisting motion, place a sterile dressing over the site and apply pressure for several minutes until the bleeding stops. Once hemostasis is achieved, a bandage should be applied, and the patient should be instructed to lie supine for 10 or more minutes. Pressure dressings may be required in thrombocytopenic patients.
• Once the biopsy needle has been removed, the specimen may be extracted from the needle by inserting the obturator (or stylet) through the distal (cutting) end of the needle. The bone marrow biopsy can then be placed on a slide, where imprints (touch prints) are made before the core specimen is further processed for cytologic investigations. This step is especially useful in situations where a bone marrow aspirate could not be obtained (Fig. 8B).
• Examine the biopsy specimen. If the specimen consists mostly of homogeneous, white material (cortical bone) or glistening tissue (cartilage), it may be necessary to attempt a second biopsy for a more satisfactory specimen. This should be done with a new biopsy needle, as the original needle may have been damaged by the process of inserting the obturator or stylet through the distal end of the biopsy needle.
Special Consideration
Precautions to be taken when dealing with a particular disease for example, in patients with multiple myeloma, the bone may be extremely soft and if one applies a normal force, the needle may pierce the entire blade of bone to injure underling structures. Also, it is very difficult to get a biopsy from soft and fragile bone. To take a biopsy from such cases, you need to palpate the bone by the needle and chose a firm to hard point on the surface of bone. Dry tap, where one is unable to get aspirate, is more commonly due to faulty technique but occasionally associated with certain underlying conditions like marrow fibrosis, tumour infiltrates, leukaemia and aplastic anaemia.
POST-PROCEDURE CARE OF BONE MARROW ASPIRATION AND BIOPSY
• Document vitals during procedure, post-procedure recovery and hemostasis
• If the patient is in pain after the procedure, give him some analgesia (e.g.paracetamol).
• Firm pressure and dressing. Advise patient to rest for at least 30 minutes to be sure about his vitals and also the hemostasis at the biopsy site. Keep the area dry. Bandage can be removed after 24 hours. Wound site should be checked to look for signs of infection or hematoma formation.
• Preparation of bone marrow slides: Put few drops of the aspirate on each glass slide and transfer the remaining aspirate into EDTA tubes to prevent clotting. Slides can be prepared by following methods.
– Wedge method: Place few aspirate particles on one slide and use the second slide at an angle of 30 degree to spread it.
– Particle crush technique: This is a better technique but requires practice. Particles are put over the end of the first slide and the second slide is placed parallel to the first one to crush the particles and then run over. This method gives better evaluation of hematopoietic cells and mast cells.
– Touch (imprint) smear: When aspirate is unsatisfactory. Biopsy piece is rolled in between two slides with mild compression making sure not to break the biopsy sample.
COMPLICATION OF BONE MARROW ASPIRATION AND BIOPSY
Complication rate is reported to be 0.05–0.07% in large studies.
• Dizziness and headache under influence of a local anesthetic agent
• Local site infection/abscess formation
• Bleeding or oozing from puncture site, hematoma.