Collection, Processing, and Examination of Bone Marrow Specimens

Meaningful interpretation of a bone marrow specimen requires an adequate and well-prepared sample. The definition of adequacy depends on the clinical indication for the examination. For example, for staging lymphoma, a bilateral bone marrow core biopsy is superior to a unilateral biopsy ; thus, for this purpose, a bilateral biopsy defines adequacy. In contrast, for the diagnosis of acute leukemia, a unilateral bone marrow aspiration and core biopsy are usually sufficient, in conjunction with appropriate immunophenotyping and genetic studies. This chapter outlines what constitutes an adequate bone marrow specimen, how to collect such a specimen, and how to process it to ensure optimal interpretation.

Medical Indications for Bone Marrow Examination

In general, a bone marrow examination is called for when there are hematologic abnormalities that cannot be explained by available clinical and laboratory data. A blood smear should always be carefully evaluated before deciding whether a bone marrow examination is necessary. For instance, circulating blasts in and of themselves do not necessitate a bone marrow evaluation if the patient has recently been treated with granulocyte colony-stimulating factor and the blood shows a dramatic neutrophilic left shift that manifests as circulating neutrophilic myelocytes and promyelocytes. Should the blasts persist despite the resolution of other neutrophilic precursors, a bone marrow examination should be considered. Aside from the diagnostic purposes outlined in Box 3-1 , staging for metastatic disease and monitoring therapy for antineoplastic effects and/or hematologic toxicity constitute the other two broad medical indications for bone marrow evaluation.

Box 3-1

Indications for Bone Marrow Examination

Diagnostic Purposes

Unexplained cytopenia or cytosis
Diagnosis of hematopoietic and lymphoid neoplasms
Workup of unexplained blasts or other abnormal cells in the blood suggestive of bone marrow pathology
Evaluation of mastocytosis, amyloidosis, and metabolic storage disorders
Workup of monoclonal gammopathy
Workup of fever of unknown origin
Workup of unexplained splenomegaly or other organomegaly

Staging for Malignant Disease

Staging of malignant lymphoma
Detection of metastatic tumor, in particular small cell tumors of childhood

Monitoring

Follow-up after induction of chemotherapy for acute leukemia and, less often, before and during consolidation or maintenance chemotherapy
Re-staging after treatment for lymphoma
Follow-up after hematopoietic stem cell transplantation
Follow-up in patients with aplastic anemia, Fanconi anemia, paroxysmal nocturnal hemoglobinuria, and other bone marrow failure syndromes to monitor for the possible development of myelodysplastic syndromes
Monitoring toxicity and antineoplastic effects of antineoplastic regimens

It has been suggested that if the blood has a sufficient quantity of blasts to meet the definition of acute leukemia and to allow other ancillary studies such as cytochemical stains, cytogenetics, and flow cytometry immunophenotyping, a bone marrow examination is superfluous. This approach may save time and money, and it may spare the patient discomfort and the risk associated with an invasive procedure. It should be noted, however, that the marrow is usually examined after the induction of chemotherapy to assess response to therapy. Such follow-up requires knowledge of the preinduction marrow landscape with its blast proportion and is not possible if only the blood has been examined at diagnosis.

Comorbid conditions such as coagulopathy, infection in close proximity to the biopsy site, or prior radiation to the biopsy site should be carefully assessed before embarking on a bone marrow biopsy. These factors are not necessarily contraindications to biopsy, and often the procedure can be modified to accommodate these circumstances. Factor replacement or reversal of anticoagulant therapy may be implemented in the case of severe coagulopathy. In the case of infected skin overlying the crests or prior radiation to the posterior iliac crests resulting in persistent marrow hypocellularity in the involved fields, the sternum may be selected for bone marrow aspiration. When the sternum is selected for marrow evaluation, obtaining core biopsies is not feasible. Of note, thrombocytopenia itself is a relatively common indication for bone marrow examination; it is usually not a contraindication for bone marrow aspiration and core biopsy as long as pressure is meticulously applied to attain hemostasis afterward. Thus, when a bone marrow examination is truly justified, the aspiration and biopsy procedure can usually be accomplished safely.

Components of a Bone Marrow Evaluation

Bone Marrow Aspiration or Trephine Biopsy

Once it has been decided that a bone marrow examination is indicated, it is important to determine what specimens to collect. Studies by Brynes and Barekman and their respective colleagues have established that a thorough bone marrow examination includes both marrow aspiration and trephine biopsy. In their review of more than 4000 diagnostic bone marrow specimens over a 10-year period at a single institution, Barekman and colleagues reported that approximately 30% of carcinomas would have been missed if the pathologist had examined only the aspirate. Conversely, in 9% of bone marrow specimens positive for metastatic carcinoma, the diagnosis was made on the aspirate alone.

The benefit of examining both the marrow aspirate and core biopsy extends beyond the evaluation of focal processes; it also applies to the workup of pancytopenia. Imbert and coworkers retrospectively examined 213 bone marrow specimens obtained over approximately 4 years at a large tertiary hospital for the evaluation of pancytopenia; “focal” processes such as lymphoma and metastatic tumor accounted for approximately 20% of the final diagnoses. Of the 213 specimens, the authors found that bone marrow aspiration alone was sufficient for diagnosis in 55% of cases; in 27%, a trephine biopsy was necessary for diagnosis. With respect to acute leukemia in which a bone marrow aspiration alone may be presumed to be sufficient, Barekman and colleagues reported positive findings in the biopsy but not in the aspirate in 20 of 576 marrow specimens obtained as follow-up for acute leukemia. Occasionally, despite a marrow “packed” with leukemic blasts at diagnosis, the aspirate may be sparsely cellular, and the diagnosis and phenotypic characterization of the leukemic blast population rest on the trephine biopsy. Taken together, these data indicate the justification for performing both marrow aspiration and core biopsy.

Bilateral or Unilateral Specimen

In general, a bilateral biopsy is recommended for maximizing sampling and enhancing the rate of detecting marrow involvement by lymphoma, metastatic tumor, or other focal infiltrative processes. Confirming earlier results by the Brunning and Juneja groups, Barekman and colleagues reported that 32% of carcinomas and 23% of lymphomas were positive on only one side. Because the aspirate is likewise subject to the artifact of focal sampling, bilateral aspiration may also be considered, especially for staging (non-epithelial) small cell tumors of childhood. It is worth noting, however, that opinions differ at both ends of the spectrum, with some proposing that if a core specimen of more than 1.6 cm in length has already been obtained, the gain in diagnostic information from additional core biopsies may not justify patient discomfort, whereas others advocate for the so-called double-bilateral bone marrow biopsy in which two core specimens are obtained from each iliac crest.

How Many Specimen Aliquots for Ancillary Studies?

In addition to obtaining aspirate and trephine biopsy samples for morphologic examination, careful consideration should be given to procuring sufficient samples for other studies as necessary for an accurate diagnosis or prognosis. In general, if the differential diagnosis includes malignancy, aspirate samples should be obtained for cytogenetic and molecular genetic analyses. If there is a possibility of acute leukemia or a lymphoid neoplasm, a sample for flow cytometry immunophenotyping analysis should also be procured. These suggestions are in accordance with the World Health Organization's recommendation to use “all available information—morphology, immunophenotype, genetic features, and clinical features—to define diseases. Last, samples for bacterial, mycobacterial, fungal, or viral cultures should be collected if an infectious cause is suspected. If the preoperative differential diagnosis is broad, extra anticoagulated aspirates should be obtained; given the increasingly greater role of molecular testing in a wide variety of hematopoietic and lymphoid disorders, considerations should be given to select the type of anticoagulant that will be most amenable to such subsequent laboratory analyses.

Collection of Bone Marrow Aspirate and Core Biopsy

Anatomic Sites

In both adults and children, the crest of the posterior superior iliac spine is preferred because of its relative distance from other vital structures and its relatively large surface area, which allows the maneuvering of biopsy and aspiration needles. An alternative site in adults is the sternum, but only marrow aspiration should be performed in this location, and only by an experienced operator; core biopsies are not done at the sternum. The anterior superior iliac spine is rarely used because of its proximity to other vital structures and because its crest is narrow. In very young children, the anterior tibial plateau can be used. Sites within previous fields of radiation should be avoided because irradiation-induced hypocellularity may persist for years.

Collection Procedures

Some authorities recommend that the trephine biopsy be obtained first. Through the same skin incision, a separate needle for aspiration is then placed and redirected through a separate bone puncture 0.5 cm to 1 cm away from the biopsy site. This sequence minimizes the morphologic distortion that can occur from interstitial hemorrhage when the aspirate is obtained first. Other authors suggest that the order of aspiration and trephine biopsy is not important as long as each sample is obtained through a separate bone puncture and with a separate and appropriate needle.

Detailed instructions on how to perform the bone marrow core biopsy and aspiration are beyond the scope of this chapter. More important, informed patient consent must be obtained after a careful discussion of the risks and benefits, and the novice should have direct personal supervision during the procedure. The following discussion focuses on aspects of the procurement procedure that are relevant to the handling of specimens.

General Approach

Because sterile technique minimizes infectious complications, it is worthwhile to work in parallel with a trained medical technician or technologist who can assist with the handling and disposition of the aspirates, cores, and instruments. Once the procedure begins, it is important to proceed quickly and efficiently to minimize patient discomfort and the clotting of specimens. As mentioned, the types of tissue obtained depend on the preoperative differential diagnosis. It is important to plan in advance the number of core biopsies and aspirate volumes, the types of anticoagulants required for all necessary laboratory testing, and, because each successive aspirate becomes more hemodiluted, the sequence in which the various aspirate samples will be obtained. It is helpful to review this sequence with the technical assistant before the procedure. It is generally recommended that the very first aspirate be reserved for morphology ; this aspirate sample should constitute no more than 1 mL to minimize any hemodilution and be without any anticoagulant. If smears for morphologic examination cannot be prepared immediately at this point, though, the aspirated marrow can be placed in ethylenediaminetetraacetic acid (EDTA), with the caution that prolonged exposure to EDTA for over 2 hours can cause morphologic distortions . EDTA is also a suitable anticoagulant for flow cytometry immunophenotyping and molecular genetic testing, whereas preservative-free heparin is preferable for cytogenetic analysis. Other reagents such as acid citrate dextrose and sodium heparin are acceptable for flow cytometry immunophenotyping analysis. It is important that the individual performing the aspiration and core biopsy procedure knows the specimen requirements of the specialty laboratories so that the correct anticoagulant is used.

Bone Marrow Trephine Biopsy Procedure and Touch Imprints

Versions of the original Jamshidi biopsy needle for procuring the core biopsy are available in both disposable and reusable forms. Most adult patients require a 4-inch, 11-gauge needle. When patients are osteopenic, a larger bore needle (8-gauge) allows the collection of an intact core biopsy with minimal crush artifact. For pediatric patients, a 2- or 4-inch, 13-gauge biopsy needle is used. Sola and associates described a bone marrow biopsy technique for neonates in which a -inch, 19-gauge Osgood needle is used.

With the exception of young pediatric patients, an adequate core biopsy, prior to fixation, should be at least 1.6 cm to 2 cm long (exclusive of cortical bone, cartilage, or periosteum) and free of crush artifact or fragmentation ( Fig. 3-1 ). Grossly, cores of marrow have a finely mottled, deep red color and a gritty texture; when the marrow is severely hypoplastic, the core may appear pale yellow, but its surface should still be gritty. Marrow that is completely replaced by leukemia, lymphoma, or other neoplasms may appear white. Cortical bone often has an ivory color with a hard, smooth surface; cartilage is gray-white with a glistening surface—findings that should prompt the operator to try again.

Figure 3-1, Example of an excellent core biopsy (>1 cm long) consisting of mostly marrow, with very little cortical bone or periosteal soft tissue (arrowhead) and with minimal crush artifact or hemorrhage. To fit these parameters, one end of this long core biopsy had to be truncated (right side).

If an adequate aspirate has not been possible, considerations should be given to preparing touch imprints of the core biopsy prior to placing it in fixative; otherwise the core biopsy should be placed in fixative immediately. There are several ways to prepare touch imprints. First, the core can be gently blotted to remove adherent blood, and several clean glass slides are then touched gently to the marrow core. One can also touch the cores to the glass slides, although this approach requires a steady hand to avoid crushing or dropping the specimen. Alternatively, the core is gently rolled between two glass slides; although this method may yield more cells on the imprints, there is also a greater risk for fragmentation of the core.

Bone Marrow Aspiration Procedure

An Illinois aspiration needle or its variant is used to collect the bone marrow aspirate. Although the needle is advanced through the same skin incision used for the biopsy, the point of puncture through the bone should be separate from the puncture site of the trephine biopsy, preferably approximately 0.5 to 1 cm away. Otherwise, the aspirate may consist of only clotted blood or marrow. Because each successive aspiration becomes more hemodiluted, a rapid and forceful aspiration of approximately 0.5 to 1 mL of fluid marrow should be obtained first for morphologic examination. Additional aspirate samples can be obtained for flow cytometry immunophenotyping analysis, cytogenetics, molecular diagnostic evaluation, and cultures, as needed and in that sequence. (In rare cases in which electron microscopic studies are called for, that sample should be collected after the initial aliquot for morphology but before that obtained for flow cytometry.) The syringes used for samples obtained for morphologic examination and electron microscopy should be free from anticoagulants; the syringes used for other studies should be coated in advance with the appropriate anticoagulants. Undiluted marrow aspirate is deep red and slightly thicker than blood. Because marrow aspiration can create intense discomfort, patients should be warned in advance, and the aspiration should be done as quickly as possible.

Processing of Marrow Trephine Biopsy and Aspirate

Trephine Biopsy

The following discussion applies to paraffin embedding. For plastic embedding, the reader is referred to several authoritative reports on the topic.

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