BONE MARROW COLLECTION AND PREPARATION

Bone marrow aspiration provides dense particles of hematopoietic cells, fat cells, and endothelial cells for morphologic evaluation. Biopsy specimens provide information on the cellularity and architecture of the marrow. Both aspiration and biopsy are almost always performed together because the information provided by the two procedures is complementary. A diagnosis can be made more often and more easily if both procedures are performed.

The site most commonly used in the adult is the posterior iliac crest. Other sites that may be used include the sternum (patients older than 15 years), anterior iliac crest (infants and children), and tibia (infants less than 18 months). The sternum and tibia are used for aspiration only.

NOTE: If the purpose of the procedure is to look for solid tumors such as lymphoma or metastatic carcinoma, bilateral iliac crest biopsies SHOULD be considered to increase the chances of detection.

BONE MARROW ASPIRATION

  1. After the patient is prepped and draped for a sterile procedure, the skin and soft tissues are injected with a local anesthetic, such as lidocaine, at the site to be sampled.
  2. Draw a small amount (1-2 cc) of 10% heparin into the aspirate syringe. Wet the interior of the syringe and expel all heparin. Ten or 20 ml syringes are better than smaller sizes because they have a stronger pull.

    a) To make 10% heparin:
    1. 1.0 cc (ml) of liquid heparin (1,000 USP units) added to;
    2. 9.0 cc (ml) of sterile physiologic saline
    3. (OR proportional amounts, according to volume of 10% heparin desired.)
  3. Make a small 3 mm slit in the skin with a scalpel blade.
  4. Insert the aspirate needle into the posterior superior iliac crest (or alternate site). Once the needle has entered the marrow cavity, the stylet is removed. A widely used needle is the University of Illinois sternal needle. Some physicians prefer to use the Jamshidi bone marrow biopsy needle. However, clotted specimens are more common if obtained through a biopsy needle.
  5. Attach the heparinized syringe, then quickly aspirate no more than 2-3 cc of bone marrow contents. More volume than this dilutes the marrow with sinusoidal blood. Immediately remove the syringe from the needle and place in EDTA (purple top) tube and invert several times. Slides may be immediately prepared at the bedside or the EDTA tube may be sent directly to WPM for processing. If additional marrow is required, another syringe is attached and the necessary volume is obtained.
  6. After aspirations are completed, remove the aspiration needle, and if desired, insert the bone marrow biopsy needle into the bone marrow space. Proceed to “BONE MARROW BIOPSY ”, at bottom of this page.
  7. If bone marrow procurement is complete after the aspiration procedure, direct pressure with a sterile gauze is applied to prevent bleeding and a small sterile adhesive bandage is placed over the needle entry site.

BONE MARROW ASPIRATE SAMPLE PREPARATION

Bone marrow in an EDTA (purple top) tube may be sent directly to WPM for aspirate slide preparation.

If on-site aspirate preparation is desired, slides must be immediately prepared from the aspirate. Squash or crush preparations are preferred. See procedure below:

  1. At the bedside, before the marrow has clotted, the person making the slides should expel a small amount of marrow aspirate into a petri dish or similar dish. Since the marrow aspiration contains a mixture of blood and marrow it is helpful to tilt the dish at a slight angle allowing the blood to flow to the bottom edge of the dish. This allows the marrow spicules to be more easily visualized. (Spicules are particles of distinct, solid tissue that are 1 mm or less in dimension.) Pick up the spicules with a Pasteur pipette, along with a small drop of blood, and place near one end of a glass microscope slide. Carefully, place another glass microscope slide on top of the other slide and gently pull them apart in a direction parallel to their surfaces.

    NOTE: As the marrow is being spread, the appearance of fat as irregular holes in the films gives assurance that marrow (not peripheral blood) has been obtained.
  2. Prepare at least 4 to 6 slides in this manner.
  3. Label all slides appropriately (at least the patient's first and last name).
  4. Place the remainder of the marrow in AZF fixative (acetic acid, zinc, formalin), label appropriately (at least the patient's first and last name), and transport to WPM Pathology Laboratory with the slides and patient information.

NOTE: If it is not possible to make the smears at the bedside, place the marrow into an EDTA (purple top) tube. Return to laboratory, place the marrow into a petri dish and make smears as directed above.

BONE MARROW FOR CYTOGENETIC (CHROMOSOME) STUDIES

Malignant hematopoietic diseases such as leukemia and lymphoma, and also myelodysplasia, result from the expansion of an abnormal clone of cells in the bone marrow. These clones can be identified by abnormal chromosomes which are detected by cytogenetic studies.

  1. Immediately after the marrow is aspirated for microscopic smears, use another syringe to aspirate approx. 3-5 ml of bone marrow aspirate and place in a sterile sodium heparin (green top) tube. Mix well by inversion.
  2. Label the tube appropriately (at least the patient's first and last name).
  3. Transport to WPM Pathology Laboratory, along with other bone marrow specimens, and a properly filled out WPM SURGICAL PATHOLOGY and NON-GYN CYTOLOGY request form requesting: “Bone Marrow Cytogenetics”.

BONE MARROW FOR FLOW CYTOMETRY

Flow cytometry is most commonly used to identify surface molecules expressed on bone marrow cells. With flow cytometry, a clonal population of cells as found in leukemias or lymphomas can be both defined and quantitated.

  1. Immediately after the marrow is aspirated for microscopic smears, use another syringe to aspirate approx. 3-5 ml of bone marrow aspirate and place in a sterile sodium heparin (green top) tube. Mix well by inversion.
  2. Label the tube appropriately (at least the patient's first and last name).
  3. Transport to WPM Pathology Laboratory, along with other bone marrow specimens, and a properly filled out WPM SURGICAL PATHOLOGY and NON-GYN CYTOLOGY request form. Under Special Requests: mark [X] Flow Cytometry.

BONE MARROW BIOPSY

  1. The bone marrow biopsy should be obtained in the usual manner, preferably from the posterior superior iliac crest. It is preferable to use a specimen capture device system, such as the Snarecoil or Core-Lock bone marrow biopsy needle systems, that will capture long, non-fragmented specimens so there will be reduced trauma to the biopsy specimen. Follow the specific biopsy system directions for use. The bone marrow biopsy specimen should be at least 2 cm in length.

    Alternatively, the Jamshidi bone marrow biopsy needle can be used. Advance the Jamshidi needle with the stylet in place until outer cortical bone is penetrated; then remove the stylet and advance the needle to obtain a marrow core specimen. The bone marrow core specimen should be at least 2 cm in length. If it is not, additional passes should be attempted after repositioning the biopsy needle. Rotate the needle completely several times. Then remove the needle, with the core specimen in its tip. Push the core backward from the tip through the needle handle using a specific probe designed for this purpose, onto a piece of gauze or directly into a container of AZF fixative.
  2. Upon completion of bone marrow procurement, apply direct pressure with a sterile gauze to prevent bleeding. Place a small sterile adhesive bandage over the needle entry site.
  3. Place the biopsy specimen in AZF fixative, label appropriately (at least the patient's first and last name), and transport to WPM Pathology Laboratory.

NOTE: If the purpose of the procedure is to look for solid tumors such as lymphoma or metastatic carcinoma, bilateral iliac crest biopsies should be considered to increase the chances of detection.

SPECIMEN TRANSPORT

The aspirate slides, aspirate specimen, and bone marrow biopsy should always be accompanied by:

  1. Concurrent peripheral blood smears (4-6).
  2. Laboratory data, especially a recent CBC and retic. count.
  3. A complete clinical history.
  4. A properly filled out WPM SURGICAL PATHOLOGY and NON-GYN CYTOLOGY request form requesting all tests desired to be performed on the bone marrow specimens.

NOTE: Formalin/formaldehyde exposure has been shown to have a detrimental effect on peripheral blood and bone marrow smears. The vapors cause the smears to not stain adequately. Therefore, we request that peripheral blood and bone marrow smears be submitted in one biohazard bag and the bone marrow aspirate and biopsy and/or any other specimens in formalin/formaldehyde in another separate biohazard bag.

For further questions or information, call a WPM pathology Laboratory Pathologist at 785-823-7201 or 800-365-3430.

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  • 338 N. Front St.
  • Salina, KS 67401
  • 785.823.7201