Bone marrow examination | A Wright's stained bone marrow aspirate smear from a patient with leukemia. |
Bone marrow examination refers to the pathologic analysis of samples of bone marrow obtained by bone marrow biopsy (often called a trephine biopsy) and bone marrow aspiration. A bone marrow aspiration should be performed as part of the same procedure. For patient safety and convenience, biopsies are usually performed on the posterior iliac crest. The biopsy specimen should measure at least 1.6 cm and, if it does not, consideration should be given to repeating the procedure, possibly on the contralateral iliac crest. Bone marrow examination is used in the diagnosis of a number of conditions, including leukemia, multiple myeloma, lymphoma, anemia, and pancytopenia. The bone marrow produces the cellular elements of the blood, including platelets, red blood cells and white blood cells. While much information can be gleaned by testing the blood itself (drawn from a vein by phlebotomy), it is sometimes necessary to examine the source of the blood cells in the bone marrow to obtain more information on hematopoiesis; this is the role of bone marrow aspiration and biopsy.
{ A trephine (/trɨˈfaɪn/; from Latin trypan, meaning to bore) is a surgical instrument with a cylindrical blade. It can be of one of several dimensions and designs depending on what it is going to be used for. They may be specially designed for obtaining a cylindrically shaped core of bone that can be used for tests and bone studies, cutting holes in bones (i.e., the skull) or for cutting out a round piece of the cornea for eye surgery.
A cylindrically shaped core of bone (or bone biopsy) obtained with a bone marrow trephine is usually examined in the histopathology department of a hospital under a microscope. It shows the pattern and cellularity of the bone marrow as it lay in the bone and is a useful diagnostic tool in certain circumstances such as bone marrow cancer and leukemia.}
Components of the procedure
Section of bone marrow core biopsy as seen under the microscope (stained with H&E).

Bone marrow aspirate.

A volunteer donating bone marrow for scientific research.
Collection Site
The safe and preferred sites for bone marrow aspiration and/or biopsy are described below.
Aspiration and biopsy * Posterior superior iliac crest: This is the most commonly employed site for reasons of safety, a decreased risk of pain, and accessibility. The posterior superior iliac crest site is localized to the central crest area. See the image below. Patient position (posterior superior iliac crest). * Anterior superior iliac crest: This is an alternative site when the posterior iliac crest is unapproachable or not available due to infection, injury, or morbid obesity. The anterior superior iliac crest site is localized to the center prominence, under the lip of the crest. This location is generally not preferred due to the dense cortical layer, which makes obtaining samples more difficult and smaller in size, as well as creates a risk for an increased painful event.
Aspiration only * The sternum is sampled only as a last resort in those older than 12 years and in those who are morbidly obese, but it should be avoided in highly agitated patients. To decrease the risk of penetrating the underlying soft-tissue organs, the sternal site is limited to a region that spans between the second and third intercostal spaces. * The tibia is sampled only for infants younger than 1 year, and the procedure is conducted under general anesthesia. This site is localized to the proximal anteromedial surface, below the tibial tubercle. The tibial location is not utilized in older patients because the marrow cellularity is not consistent.
Procedure
Aspiration sampling is generally performed before marrow biopsy of the posterior/anterior iliac crest due to the fact the biopsy technique induces elevated thromboplastic substances. The consequence of this is a reduction in the effectiveness of an aspiration sampling.[6, 10, 11]
Aspiration
* The patient is placed in the lateral decubitus position, with the top leg flexed and the lower leg straight. Alternatively, the patient may be placed in the prone position. * Palpate the iliac crest, and mark the preferred sampling site with a pen. * Aseptic technique is employed, including sterile gloves and gown. * The site is prepared with an antiseptic (eg, povidone-iodine or chlorhexidine gluconate), scrubbed, and draped, exposing only the site to be sampled. See the images below. Skin preparation. Site preparation. * The skin and the underlying tissue to the periosteum are infiltrated with a local anesthetic (eg, approximately 10 mL of 1% Xylocaine [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. Local anesthetic injection. * 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 to not only decrease the patient's anxiety level, but to minimize movements that may affect the efficacy of the procedure. Having a family member present may help to alleviate the patient's anxiety. To ensure sufficient pain control is 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. * 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. Aspiration needle placement. * 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 noted by a sudden reduction in pressure. The depth of the penetration should not extend beyond an initial 1 cm. See the image below. Bone marrow aspiration. * Once within the marrow cavity, the stylet is removed. Using a 20 mL syringe, approximately 0.3 mL of bone marrow is aspirated. A volume greater than 0.3 mL may dilute 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. 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, collecting 5 mL at a time. The samples are then transferred into an anticoagulant-containing tube that is appropriate to the requested study: heparin for cytogenetic analysis; either heparin or EDTA for immunophenotyping; formalin for a Cytoblock preparation; and, gluaraldehyde for ultrastructural examination. * The marrow needle is removed, and pressure is applied to the aspiration site with gauze until any bleeding has stopped (see Postprocedure Care). * Once the aspiration is completed, the specimen is processed by the hematopathology technician.
Bone marrow biopsy
Any of several needle models can be utilized; however, the Jamshidi needle is considered the most popular. This disposable needle is tapered at the distal end to help retain the specimen for improved extraction. * Patient preparation is to be followed in the manner previously described for bone marrow aspiration. Some kits allow for aspiration and biopsy to be obtained from 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, aspiration artifact is created where the marrow has been aspirated out of the core. * The needle, with stylet locked in place, is held with 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. Bone marrow biopsy. Jamshidi needle placement. Bone marrow biopsy. Jamshidi needle placement. * Using firm pressure, slowly rotate the needle in an alternating clockwise-counterclockwise motion, and advance it into the bone marrow cavity to obtain an adequate bone marrow specimen measuring approximately 1.6-3 cm in length. * Rotate the needle along its axis to help loosen the sample, pull back approximately 2-3 mm, and advance the needle again slightly, at a different angle, to help secure the specimen. * Following this procedure, slowly pull the needle out, while rotating in an alternating clockwise and counterclockwise motion. * Remove the specimen from the needle and introduce a probe 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 placing the specimen in formalin. * Place the specimen in formalin solution for histologic processing. See the images below.Bone marrow biopsy specimen. Bone marrow biopsy specimen. Bone marrow biopsy specimen in fixative solution. * The marrow needle is removed, and pressure is applied to the site with gauze until any bleeding has stopped (see Postprocedure Care).
The Sternum
Note: With this site, only aspiration is to be performed, and it is only to be performed 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. Note: The area chosen should be to one side of the midline as 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 and gown. * Local anesthetic is used to infiltrate from the skin to the periosteum. * After 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 in the above section (see Procedure: Posterior/Anterior Iliac Crest), advance the needle into the marrow cavity, obtain the specimen, and remove the needle. Note: Unlike other sites, the attached guard is not to be removed; rather, it is adjusted to allow for the maximum depth of needle penetration to 0.5 cm. This prevents needle slippage that can result in injury to the underlying mediastinal organs. * Core biopsies are not to be performed from the sternum
Bone marrow samples can be obtained by aspiration and trephine biopsy. Sometimes, a bone marrow examination will include both an aspirate and a biopsy. The aspirate yields semi-liquid bone marrow, which can be examined by a pathologist under a light microscope and 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, 2mm wide and 2 cm long (80 μL), which is examined microscopically (sometimes with the aid of immunohistochemistry) for cellularity and infiltrative processes. An aspiration, using a 20 mL syringe, yields approximately 300 μL of bone marrow.A volume greater than 300 μL is not recommended, since it may dilute the sample with peripheral blood. Comparison | | Aspiration | Biopsy | Advantages | * Fast * Gives relative quantity of different cell types * Gives material to further study, e.g. molecular genetics and flow cytometry | * Gives cell and stroma constitution * Represents all cells * Explains cause of "dry tap" (aspiration gives no blood cells) | Drawbacks | Does not represent all cells | Slow processing |
Aspiration does not always represent all cells since some such as lymphoma stick to the trabecula, and would thus be missed by a simple aspiration.
Site of procedure
Bone marrow aspiration and trephine biopsy are usually performed on the back of the hipbone, or posterior iliac crest. An aspirate can also be obtained from the sternum (breastbone). For the sternal aspirate, the patient lies on his back, with a pillow under the shoulder to raise the chest. A trephine biopsy should never be performed on the sternum, due to the risk of injury to blood vessels, lungs or the heart. Bone marrow is also perform from the tibial (shinbone) site in children up to 2 years of age. Spinous process aspiration in this site usually L3 - L4 is puncture in a lumber puncture position.
How the test is performed

A needle used for bone marrow aspiration, with removable stylet.

The OnControl Bone Marrow Biopsy and Aspiration System with Aspiration Needle.
A bone marrow biopsy may be done in a health care provider's office or in a hospital. Informed consent for the procedure is typically required. The patient is asked to lie on his or her abdomen (prone position) or on his/her side (lateral decubitus position). The skin is cleansed, and a local anesthetic such as lidocaine or procaine is injected to numb the area. Patients may also be pretreated with analgesics and/or anti-anxiety medications, although this is not a routine practice.
Typically, the aspirate is performed first. An aspirate needle is inserted through the skin using manual pressure and force until it abuts the bone. Then, with a twisting motion of clinician's hand and wrist, the needle is advanced through the bony cortex (the hard outer layer of the bone) and into the marrow cavity. Once the needle is in the marrow cavity, a syringe is attached and used to aspirate ("suck out") liquid bone marrow. A twisting motion is performed during the aspiration to avoid excess content of blood in the sample, which might be the case if an excessively large sample from one single point is taken.
Subsequently, the biopsy is performed if indicated. A different, larger trephine needle is inserted and anchored in the bony cortex. The needle is then advanced with a twisting motion and rotated to obtain a solid piece of bone marrow. This piece is then removed along with the needle. The entire procedure, once preparation is complete, typically takes 10–15 minutes.
If several samples are taken, the needle is removed between the samples to avoid blood coagulation.
In 2010, a power system was offered for sale. Previously, needles were forced through the bone manually, which required significant upper-body strength and effort by the person performing the procedure. The power system, made of a specially designed needle and a powered driver similar to a power drill, produced comparable or better core sample quality in tests. It was also much faster and easier.
After the procedure
After the procedure is complete, the patient is typically asked to lie flat for 5–10 minutes to provide pressure over the procedure site. After that, assuming no bleeding is observed, the patient can get up and go about their normal activities. Paracetamol (aka acetaminophen) or other simple analgesics can be used to ease soreness, which is common for 2–3 days after the procedure. Any worsening pain, redness, fever, bleeding or swelling may suggest a complication. Patients are also advised to avoid washing the procedure site for at least 24 hours after the procedure is completed.
Postprocedure Care
After the procedure, firm pressure is applied 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.
The patient is to be discharged with orders that the wound dressing is to 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, these findings are to be reported to the clinician’s office.
Contraindications
There are few contraindications to bone marrow examination. The only absolute reason to avoid performing a bone marrow examination is the presence of a severe bleeding disorder which may lead to serious bleeding after the procedure. If there is a skin or soft tissue infection over the hip, a different site should be chosen for bone marrow examination. Bone marrow aspiration and biopsy can be safely performed even in the setting of extreme thrombocytopenia (low platelet count).
Complications
While mild soreness lasting 12–24 hours is common after a bone marrow examination, serious complications are extremely rare. In a large review, an estimated 55,000 bone marrow examinations were performed, with 26 serious adverse events (0.05%), including one fatality.The same author collected data on over 19,000 bone marrow examinations performed in the United Kingdom in 2003, and found 16 adverse events (0.08% of total procedures), the most common of which was bleeding. In this report, complications, while rare, were serious in individual cases.

Giemsa stain

Giemsa stained Trypanosoma parasites (Chagas disease pathogen)

Whirling disease section stained with Giemsa
Giemsa stain, named after Gustav Giemsa, an early German microbiologist, is used in cytogenetics and for the histopathological diagnosis of malaria and other parasites.[1] |
Uses
It is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. Giemsa stain is used in Giemsa banding, commonly called G-banding, to stain chromosomes and often used to create an idiogram. It can identify chromosomal aberrations such as translocations and rearrangements.
Giemsa stain is also a differential stain. It can be used to study the adherence of pathogenic bacteria to human cells. It differentially stains human and bacterial cells purple and pink respectively. It can be used for histopathological diagnosis of malaria and some other spirochete and protozoan blood parasites. It is also used in Wolbach's tissue stain.
Giemsa stain is a classic blood film stain for peripheral blood smears and bone marrow specimens. Erythrocytes stain pink, platelets show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta.
Giemsa stain is also used to visualize chromosomes.
Giemsa stains the fungus histoplasma, chlamydia bacteria, and can be used to identify Mast cells.
Generation
Giemsa's solution is a mixture of methylene blue, eosin, and Azure B. The stain is usually prepared from commercially available Giemsa powder.
A thin film of the specimen on a microscope slide is fixed in pure methanol for 30 seconds, by immersing it or by putting a few drops of methanol on the slide. The slide is immersed in a freshly prepared 5% Giemsa stain solution for 20–30 minutes (in emergencies 5–10 minutes in 10% solution can be used), then flushed with tap water and left to dry.

Wright's stain
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Wright's stain is a histologic stain that facilitates the differentiation of blood cell types. It is used primarily to stain peripheral blood smears and bone marrow aspirates which are examined under a light microscope. In cytogenetics it is used to stain chromosomes to facilitate diagnosis of syndromes and diseases.
It is named for James Homer Wright, who devised the stain, a modification of the Romanowsky stain, in 1902. Because it distinguishes easily between blood cells, it became widely used for performing differential white blood cell counts, which are routinely ordered when infections are suspected.
There are related stains known as the buffered Wright stain, the Wright-Giemsa stain, and the buffered Wright-Giemsa stain, and specific instructions depend on the solutions being used, which may include Eosin Y, Azure B, and Methylene Blue (some commercial preparations combine solutions to simplify staining). The May-Grünwald stain, which produces a more intense coloration, also takes a longer time to perform.
White blood cells stained with Wright's stain:

lymphocyte

basophil

eosinophil