Before the invention of immunophenotyping, examination of the bone marrow was the required method of diagnosing CLL. By agreed definition, anyone whose lymphocytes made up more than 30% of the blood cells in the marrow was presumed to have CLL. That, together with the signs, symptoms, and a complete blood count (CBC) of the peripheral blood made the diagnosis. Bone marrow biopsies are no longer required to diagnose CLL, but biopsies and aspirates are regularly used to assess disease extent, degree of marrow involvement, effect of treatment, and readiness for transplant.

Bone marrow biopsies and aspirates look very much alike. They are drawn from the same sites; they even use the same insertion needle. They are different in the type of specimen that they withdraw. Biopsies take a solid core of marrow with all of its structures such as capillaries and collagen fibers intact. Aspirates sacrifice the architecture of the marrow in order to spread the cells out more thinly so that the individual cells can be evaluated. Some conditions can be evaluated just by biopsy; others just by aspirate. Many physicians prefer to obtain both types of specimens so that a more complete picture of the marrow can be seen. It is a difficult specimen collection, so it makes sense to get as much information as possible with a single needle.

The bone can be thought of as a commercial honeybee hive. The object is to get through the outer structure of the hive without damaging it and take some of the honeycomb and some of the honey. The marrow, which is represented as the honeycomb, is three-dimensional and contains significant numbers of support structures such as collagen, arteries, veins, capillaries, and lymphatics. The aspirate, which is the honey, contains the developing blood (hematopoietic) cells.

Bone Marrow Biopsy

In bone marrow biopsies, an extremely sharp but hollow needle-like tool is placed into the marrow and twisted. The piece of marrow inside the hollow is removed as a unit with all of its cells and structures untouched. This is the biopsy material and it is treated in the histology laboratory the same way that one treats biopsies of lymph nodes, of breast tissue, or any other tissue.

This specimen is placed into hot wax (paraffin), which permeates the entire specimen giving it strength and rigidity. It is then cut into extremely thin sections (approximately 4-6 millionths of an inch). These sections are then stained and the physician examines them under the microscope. It is then possible to see if there is any damage to the structure of the marrow. Are there, for example, increased amounts of scarring (fibrosis)? Is there damage to the vessels (arteries, veins, and capillaries) that provide the marrow with nutrients? Is there sufficient iron storage in the tissue cells? It is in this sample that one can evaluate cellularity. As one ages, the number of hematopoietic cells lessens, and the amount of fat increases. Cellularity can only be evaluated if you know for sure that you have a sample that contains all of the cells in a given volume, so cellularity is only reported on a biopsy or clot section. Overall numbers of cell types can be identified and how they are grouped together can be viewed as well.

Bone Marrow Aspirate

One of the drawbacks to a biopsy is that many of the cells cannot be seen well. This is primarily because the stain used is not specific to hematopoietic cells, but also there is the issue of the damage that is done to the cells in the treatment process. While individual cells are seen, the processing concentrates on structure and relationship, not on individual cells. To see individual cells requires a clear separation of cells. In order to accomplish this, a drop or two of liquid marrow is placed on a slide, and another slide is placed on top of it, and the specimen is squeezed between the two slides, causing it to spread out. This spreading destroys any structural components, but it provides a thin, well-separated coating of marrow across the slide. When this is stained with the usual stains found in a hematology laboratory, one can view the smear with a microscope and identify individual cells and assess their quality.

It is from the aspirate that a differential report is produced showing percentages of different cell lines. The differential section of biopsy/aspirate lab report looks similar to a complete blood count (CBC) report, but it is performed by identifying between 500 and 1000 cells instead of the traditional 100 cells used in the peripheral blood differential. The percentage of lymphocytes shown in this section of the report represents the percentage of marrow involvement that is often quoted in CLL. This percentage is a key indicator in determining the extent of disease, the efficacy of treatment, and in preparing patients for transplant. Most transplant centers want this percentage to be below ten percent prior to transplant.

Common Processes

Multiple slides of each type of specimen are evaluated and each type of material will have its place in the report. As a consequence, it may appear that the report contains the same thing over and over. It does. It is possible for a report to contain 2 different slide reports from the biopsy, 2 reports from the aspirate and reports from additional or special staining techniques. Why multiple slides? Many conditions are very focal, and appear in small discrete places. If only one slide was evaluated, the condition might be missed entirely. In some institutions, marrows may be checked by a minimum of 4 slides from each type of specimen.

Each slide is evaluated and the total impression is reported. The steps listed are performed on each slide. Different steps are performed with differing levels of magnification. For some steps, a small amount of magnification is needed; for others a very high amount of magnification is needed.

At the lowest level of magnification, the first step is to confirm that there are spicules of real bone marrow present. It is possible to get just fat deposits or just blood. Without the spicules, you cannot tell if what you are looking at is really a reflection of the marrow or just material that was picked up by the aspiration. So you will see on the report some comment about the quality of the aspirate that mentions spicules. If you or your physician sees that the specimen was poor or good, then you can interpret the results in the light of that comment.

The next step in the evaluation assesses the relative cellularity of the specimen. This is best done on the biopsied material and not the aspirate but many times there will be a confirmatory comment about cellularity on an aspirate's report. In general, as one ages, the cellularity of the marrow declines and that space is used by fat cells. Newborns have essentially no fat and 100% cellularity while an eighty year old will have approximately 60% fat and 40% blood producing cells. If the marrow is under stress and can increase the number of blood producing cells, then the cellularity will be increased. The next step is to scan the slide for the presence of any large abnormal cells or clumps of cells. Malignant cells tend to adhere to each other so they may be in abundance in one portion of the slide and not in another. The cells from which platelets arise (megakaryocytes) are also evaluated. These cells are quite large and relatively few in number so you scan the entire slide to get a sense of how many are present. It is important for megakaryocytes to be present in adequate numbers and to be seen to be producing platelets.

At the next magnification level, the Myeloid to Erythroid (M:E) ratio is performed. Since red cells live significantly longer than white blood cells, usually there are three to four times the number of white cell precursors to red cell precursors. On the slides containing biopsied material, comments about the support structure the vessels, bone cells, and collagen are made.

At the highest level of magnification, individual cells are evaluated and comments are made about their numbers and quality. At this point comments about the exact appearance of the abnormal lymphocytes are made. In CLL, commonly used words are "monotonous in appearance" or "clonal expansion". The red cell precursors are evaluated to see if there are problems with hemoglobin synthesis (most typically from iron deficiency) or if there are problems with the nucleus (most typically from folic acid deficiency) or if they are adequate in number. These comments support the CBC's hemoglobin, hematocrit, and red cell indices results. Finally, the platelet production is evaluated. By looking at megakaryocytes, one can see if they are producing platelets and if the platelets being produced are appropriate looking.

Special testing

One test that is routinely performed on bone marrow biopsy material is a test for iron deposits. This requires the use of a different stain so while it is a routine test it is usually thought of as a special stain. In this stain, iron deposits are stained blue while the cells themselves stain pink. The absence of any blue deposits confirms that the person lacks adequate iron and is either iron deficient or barely able to keep up with need. In addition to certain inherited conditions, there are a number of ways in which a person may have too much iron stored. One of the more common ways is by repeated transfusions. Another is by using too little iron because there is a suppression of red cell production since hemoglobin production accounts for over 90% of all iron in the body. Iron stores will not tell which process is involved but will provide the physician with a needed piece of information for treatment decisions.

Additionally, marrow cells can be tested by flow cytometry, cytogenetics, FISH, or PCR techniques. This is important because there are situations in which malignant cells will be found in the marrow only or in the nodes only or in the peripheral blood only. Close monitoring of where the malignant cells are is important in prognosis as well as treatment.