By Craig E. Litz, M.D.

 

In general practice, this seemingly trivial issue is the single most important element in providing your patient with the proper diagnosis. The idea is to do the bone marrow biopsy once and do it right. This can be achieved if one understands what is being done and why. The following best practices provide important information regarding bone marrow specimens for the practicing hematologist/oncologist.

1) Keep smears/imprints pure

Marrow smears and imprints do matter. Hematopathologists need a superbly prepared and stained marrow smear or marrow trephine imprint to detect subtle myelodysplastic changes and blasts. The better this material, the more likely a low-grade myelodysplastic syndrome will be detected and an accurate blast percentage provided. Unfortunately, smears or imprints are the most easily damaged. Preparation is demonstrated at www.propath.com and is surprisingly easy. Briefly, a drop of neat (non-anticoagulated) bone marrow is placed on a clean glass slide immediately after aspiration and spread with another slide into a uniform monolayer.

This slide is quickly air dried only. A blood or marrow smear or touch prep should not be sprayed with or dipped in a fixative such as hairspray, alcohol or formalin. The Romanowsky stains used in hematology will only work on air-dried material that is relatively fresh (less than one month old.

2) Protect your unstained smears from formalin

This is a corollary to rule 1. Formalin fume exposure ruins smear material. While this is generally not a problem when preparing and staining smears in the same location as the biopsy procedure, it has become a problem with specialty bone marrow labs that require the core bone biopsies in formalin bottles to be shipped in the same container with air-dried smear material. Formalin fume leakage is common during transport and must be protected against by placing the formalin bottles in a sealed plastic bag provided in the kit.

3) Provide a blood smear

Blood derives from marrow; the two are intimately and dynamically linked. It follows that a comprehensive and complete hematopathological review includes a blood smear exam.

Several key findings such as a post splenectomy blood picture, hemolysis, large granular lymphocytosis, monocytosis or basophilia will be missed if a blood smear is not sent with the marrow. This could lead to a delay in diagnosis. A blood smear is prepared from a drop of blood from a finger stick or fresh EDTA (purple top) tube of blood in a similar fashion as the marrow aspirate smear (see above). All rules regarding marrow aspirate smears hold for blood smears.

4) Provide touch preps

Can’t get an aspirate due to poor positioning of the aspirate needle or marrow fibrosis? Perform several trephine imprints on the fresh core bone biopsy before putting the core in formalin. This easy procedure is demonstrated on www.propath.com. This provides similar cytologic material as the aspirate smear; myelodysplastic changes and accurate blast counts can be performed. This is one of those pearls that are not universally known but can save your patient another bone marrow biopsy procedure. One should make this a routine procedure during the bone marrow biopsy procedure even in case of an apparently adequate aspirate specimen.

5) Dry tap? Use a piece of fresh bone core for cytogenetic, flow cytometric and molecular diagnostic tests

This is a corollary to rule 4. Many oncologists/hematologists don’t know that a fresh core specimen can be used for all of the special tests. These tests require living marrow cells which a fresh core specimen has. It is best to put the fresh core in RPMI cell culture media in a leak-proof container and immediately transport at room temperature to the lab. Call the hematopathologist at the laboratory you use and let him or her know the specimen is coming. You otherwise risk unwary lab personnel at the receiving laboratory inadvertently destroying the specimen by placing it in formalin. Note: once you put the core in formalin, the cells are instantly fixed (killed) and no longer useful for these tests.

6) 1cm is not enough, 2cm is good, 3cm is better

For certain diseases such as myelodysplastic syndromes and leukemias, the length of the biopsy is not as important. These diseases involve the marrow in a homogenous fashion. Many other diseases such as myeloma, lymphoma and metastatic tumor, however, are characterized by focal marrow involvement. In the former case, any marrow length is adequate; for diseases that involve the marrow focally at least 2cm should be obtained. To make sure that all diseases have an adequate chance of being detected in the marrow, 2-3cm is recommended (Fig.3).

7) Provide an aspirate clot

An aspirate clot specimen ends up being the only non-decalcified, formalin-fixed tissue from the bone biopsy. This provides ideal material on which to perform immunohistochemical staining. Stains that are frequently sensitive to decalcification such as CD5, CD10, ZAP70, and Cyclin D1 can be performed on this material. This can be important in the classification of lymphomas.

8) Be generous with the number of marrow smears prepared

Besides the usual stains, marrow smears can be used for cytochemical stains such as myeloperoxidase, non-specific esterase, tartrate-resistant acid phosphatase and immunostains such as ZAP70. Providing a number of marrow smears allows a backup for the particle clot and core biopsy material.

9) Yellow top tube can be used in a pinch for special tests

An Acid Citrate Dextrose anticoagulant (yellow top) tube (Fig.4) can be used for all special studies. This anticoagulant keeps cells viable and doesn’t interfere with any of the enzymes or reagents employed in molecular, flow cytometric or cytogenetic studies. If you don’t know what tube to reach for in a pinch, use the yellow top.

10) If in doubt, let hematopathologist do the special test ordering

If you are unsure about what test to order or worried about omitting a test in making the diagnosis of a hematologic disorder, check the comprehensive evaluation box on the ProPath requisition (Fig.5). This allows the hematopathologist to order all the appropriate tests for making a diagnosis based on the morphologic appearance of the marrow combined with the clinical data.

Please see our online video for more details.