Bone Marrow Aspiration and Biopsy Technique

Aspiration sampling is generally performed before marrow biopsy of the posterior or anterior iliac crest. The reason is that the biopsy technique induces elevated thromboplastic substances, and this leads to a reduction in the effectiveness of an aspiration sampling. ^{[3, 18, 19]}

With the patient positioned as previously described (see Patient Preparation), the iliac crest is palpated and the preferred sampling site marked with a pen. If the patient is obese, it is helpful to ask him or her to place a hand on the hip so as to facilitate identification of the pelvic rim. It is not uncommon for the lateral sacral crest to be mistaken for the posterior superior iliac crest, an error that will lead to a painful procedure and a dry tap.

Aseptic technique is employed, including sterile gloves. The site is prepared with an antiseptic (eg, povidone-iodine or chlorhexidine gluconate), scrubbed, and draped so that only the area immediately surrounding the site to be sampled is exposed. (See the images below.).

The skin and the underlying tissue to the periosteum are infiltrated with a local anesthetic (eg, ~10 mL of 1% lidocaine). A 10-mL syringe with a 25-gauge needle is used to inject an initial 0.5 mL directly under the skin, raising a wheal. A 22-gauge needle is used to penetrate deeper into the subcutaneous tissue and the underlying periosteum, an area roughly 1 cm in diameter. (See the image below.)

The adequacy of the anesthesia is tested by gently prodding the periosteum with the tip of the needle and questioning the patient for any painful sensation. It is important to be aware of changes in the patient's comfort level throughout the procedure, not only to decrease the patient's anxiety level but also to minimize movements that may affect the efficacy of the procedure.

Having a family member present may help alleviate the patient's anxiety. To ensure that pain control is sufficient and being managed well, the person performing the procedure should talk to the patient, discuss the steps taken throughout the process, and listen to the manner as well as the content of the patient's response. The author often advises patients to concentrate on their breathing, inspiring slowly through the nose and expiring slowly through an open mouth; this helps ease any anxiety and pain.

Imaging guidance in the form of computed tomography (CT) may employed to faciliatate the procedure; fluoroscopic guidance may be an option in settings where the availability of CT may be limited. ^[20]

A skin incision is made with a small surgical blade, through which the bone marrow aspiration needle, with a stylet locked in place, is inserted. (See the image below.)

Once the needle contacts the bone, it is advanced by slowly rotating clockwise and counterclockwise until the cortical bone is penetrated and the marrow cavity is entered. Contact with the marrow cavity is usually signaled by a sudden reduction in pressure. The depth of the penetration should not extend beyond an initial 1 cm. (See the image below.)

Once within the marrow cavity, the stylet is removed. A 20-mL syringe is used, and approximately 0.3 mL of bone marrow is aspirated. Aspirating more than 0.3 mL risks diluting the sample with peripheral blood and thus is not recommended.

The material collected for bone marrow slides is generally not mixed with an anticoagulant, and it is processed immediately by a technologist; this avoids any cellular morphologic artifacts. (See Slide Preparation.) If there is to be a delay in slide preparation, place the sample in an EDTA (ethylenediaminetetraacetic acid) anticoagulant-containing tube, preferably a pediatric-sized tube to avoid exposure to excess anticoagulant.

If additional marrow is needed for ancillary studies, subsequent specimens are obtained by attaching a separate syringe and collecting 5 mL at a time. Usually, a 20-mL syringe with 1 mL of 1:1000 heparin is prepared before the aspiration procedure is started so that the samples do not clot easily. The samples are then transferred into an anticoagulant-containing tube that is appropriate to the requested study, as follows:

• 10-mL heparinized aspirate into heparin for cytogenetic analysis and immunophenotyping/flow cytometry
• 3-mL nonheparinized aspirate into EDTA for molecular studies involving PCR
• Formalin for a Cytoblock preparation (clot)
• Glutaraldehyde for ultrastructural examination
• If the biopsy is for workup of fever of unknown origin, sterile nonheparinized aspirates, 5 mL each, are also drawn for blood cultures for aerobes, anaerobes, acid-fast bacilli, and fungi 

The marrow needle is removed, and pressure is applied to the aspiration site with gauze until any bleeding has stopped (see Postprocedural Care).

Once the aspiration is completed, the specimen is processed by the hematopathology technician.

Any of several needle models can be utilized; however, the Jamshidi needle has been the most popular. This disposable needle is tapered at the distal end to help retain the specimen for improved extraction. Powered drill devices have also been employed for this purpose and currently appear to yield comparable results. ^[21]

Patient preparation is carried out in the manner previously described for bone marrow aspiration. Some kits allow aspiration and biopsy to be done with the same needle, which is convenient for the patient. However, if the latter is used, it is important to change the needle position slightly to a different area of bone after aspiration is obtained. Otherwise, an aspiration artifact is created where the marrow has been aspirated out of the core.

The needle, with stylet locked in place, is held within the palm and index finger and repositioned so that a new insertion site is created for biopsy sampling. Once the needle touches the bone surface, the stylet is removed. (See the images below.)

With firm pressure applied, the needle is slowly rotated in an alternating clockwise-counterclockwise motion and advanced into the bone marrow cavity to obtain an adequate bone marrow specimen measuring approximately 1.6-3 cm in length.

The needle is rotated along its axis to help loosen the sample, pulled back approximately 2-3 mm, and then advanced again slightly, at a different angle, to help secure the specimen. After this procedure, the needle is slowly pulled out while being rotated in an alternating clockwise and counterclockwise motion.

The specimen is removed from the needle, and a probe is introduced through the distal cutting end. If the aspirate was unsuccessful (ie, a dry tap), the core biopsy may be used to make touch preparations (see Slide Preparation). This must be performed before the specimen is placed in formalin. Finally, the specimen is placed in formalin solution for histologic processing. (See the images below.)

The marrow needle is removed, and pressure is applied to the site with gauze until any bleeding has stopped (see Postprocedural Care).

At this site, only aspiration is to be performed, and it is to be performed only on adolescent and adult patient populations.

The second to third intercostal level of the sternum is palpated, and the selected sample site is marked with a pen. The area chosen should be to one side of the midline because the marrow cellularity is considered to be diminished at that location.

The designated area is prepared with an antiseptic scrub and draped. Aseptic technique is employed, including sterile gloves. Local anesthetic is used to infiltrate from the skin to the periosteum.

After a small cut is made in the skin with a surgical blade, the aspiration needle, with the stylet locked in place, is inserted until the needle touches the bone. With the same technique described previously, the needle is advanced into the marrow cavity, the specimen obtained, and the needle then removed. At this site, unlike other sites, the attached guard is not to be removed; rather, it is adjusted to limit the maximum depth of needle penetration to 0.5 cm. This prevents needle slippage that can result in injury to the underlying mediastinal organs.

Again, core biopsies are not to be performed from the sternum.

After the procedure, apply firm pressure for 5 minutes to several layers of sterile gauze placed over the wound site. Remove residual antiseptic to avoid further skin irritation by the solution.

If hemorrhage from the wound persists, then place the patient in the supine position, with gauze over the wound site, so that consistent pressure can be applied for a minimum of 30 minutes. Rarely, bleeding may be present; if that is the case, consider placing a pressure dressing, again with the patient in a supine position, for an additional 1 hour. ^[18]

Discharge the patient with orders that the wound dressing be maintained in a dry state for 48 hours. The wound site is to be checked frequently, and if persistent bleeding or worsening pain occurs, the patient must report these findings to the clinician’s office.

This stage in bone marrow preparation should be performed by trained personnel (eg, a hematopathology technician). Thin-spread preparations of aspiration-collected samples, placed onto glass slides, can be prepared by numerous methods, all of which are aimed at retaining and evaluating marrow particles. These spicules of fat droplets (not prominently seen in pediatric cases) and fragmented bone are likely to have adherent cellular material and thus to be a target for morphologic evaluation. (See the images below.)

An aspirate smear (or wedge) is the simplest of the methods, similar in presentation to a peripheral blood smear. A drop of the acquired specimen is placed 1 cm from the edge that opposes the frosted "labeled" end, and with a second glass slide placed at a 30º angle, the sample is pushed toward the opposing side in one rapid smooth stroke. Excess sample can be removed by tilting the glass slide onto gauze or pipetting the extraneous fluid.

Squash (or crush) preparations are prepared on glass slides by placing marrow particles on a slide and pressing the particles with another slide. These preparations allow better observation of cellular interactions, in that they preserve the architecture of the marrow unit.

One report found that the crush technique is better for evaluating the percentage composition of bone marrow cells, whereas the wedge technique may be better for identifying cellularity. ^[22]  A comparative study comparing the squash and wedge techniques found the two to be diagnostically concordant in 91.7% of cases (188/205), with major discrepancies in 3.9% (8/205) and minor ones in 4.4% (9/205). ^[23] In this study, squash smears significantly overestimated bone marrow cellularity and megakaryocyte numbers were underestimated, with no differences in myeloid-to-erythroid ratio or morphology. 

The cover slip method produces more highly concentrated samples than the squash preparation does. The aspirate particles are selected from a petri dish and directly placed onto a glass cover slip. In a manner similar to the squash method, a second cover slip is gently applied to crush the sample. Each cover slip is then stained individually. Thus, enhanced removal of contaminating peripheral blood is performed, again with retention of the marrow unit architecture.

At times, biopsy touch prints are useful, especially if the aspirate is dry and the only sample available is the bone marrow biopsy. In touch preparations, the hematopathology technician gently touches the tissue fragment onto a glass slide; this can provide morphologic details similar to those that can be obtained with an aspirate.

Marrow particles can be collected in aggregate as a clot and processed in a similar manner to that of tissue. The solid component is concentrated by placing the specimen in a finely meshed bag that retains the tissue fragments but allows excess fluid to escape.

Standard stains used for the initial evaluation include Wright and May-Grunwald-Giemsa stains, which enhance cytologic detail. Other special stains can be utilized for various purposes, such as Prussian blue for iron in cases of suspected hemosiderosis or for the ringed sideroblasts of myelodysplastic syndromes. Myeloperoxidase, Sudan Black B, and leukocyte alkaline phosphatase are used in categorizing acute myeloid leukemias. Periodic acid-Schiff (PAS) stain enhances depiction of cells that are implicated in glycogen storage diseases. ^{[10, 18, 24]}

In 2002 the British Society of Haematology initiated an annual survey to assess the various types and incidence of bone marrow biopsy adverse events. ^[25]  Bain summarized results of a 7-year (1995-2001) retrospective study and identified 26 adverse events among approximately 54,890 biopsies, with an overall annual incidence of 0.05%. The most common side effects, in order of decreasing frequency, were the following:

• Hemorrhage
• Needle breakage
• Infection

Risk factors for hemorrhage included concurrent anticoagulation therapy or underlying myeloproliferative/myelodysplastic syndrome, leading to dysfunctional platelets or acquired inhibitors in the clotting cascade. Two cases were fatal and were attributed to sepsis and massive hemorrhage. ^[25]

Four years later, a prospective study by Bain revealed 15 adverse events in a single year, with an overall incidence of 0.07%, not significantly different from the previous study's results. ^[19]  However, although hemorrhage was still considered the most commonly encountered side effect, pain, anaphylactic reaction, and fractures were found to be prominent secondary consequences. Two fatalities, attributed to laceration of blood vessels, were reported from 20,323 bone marrow aspiration and biopsy procedures.